Methods and agents for improving targeting of cd138 expressing tumor cells

ABSTRACT

Disclosed are immunoconjugates having specificity for CD138 that diminish adhesion of CD138 expressing tumor cells to stroma cells and methods of using the same. This diminished adhesion renders the tumor cells not only susceptible to the immunoconjugate, but also to other agents, in particular cytotoxic agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application61/016,614, filed Dec. 26, 2007, provisional application 61/087,466,file Aug. 8, 2008 and provisional application 61/087,590, also filed onAug. 8, 2008, which are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

The present invention relates to the use of immunoconjugates against theantigen CD138 and compositions comprising the immunoconjugates todiminishing adhesion of stroma cells to CD138 expressing target cellsand thus to more effectively treat disease states involving CD138expressing cells.

BACKGROUND

CD138, which acts as a receptor for the extracellular matrix, isoverexpressed on multiple myeloma (MM) cells and has been shown toinfluence MM cell development and/or proliferation. CD138 is alsoexpressed on cells of ovarian carcinoma, kidney carcinoma, gall bladdercarcinoma, breast carcinoma, prostate cancer, lung cancer, coloncarcinoma cells and cells of Hodgkin's and non-Hodgkin's lymphomas,chronic lymphocytic leukemia (CLL) to name just a few.

The publications and other materials, including patents, used herein toillustrate the invention and, in particular, to provide additionaldetails respecting the practice are incorporated by reference. Forconvenience, the publications are referenced in the following text byauthor and date and/or are listed alphabetically by author in theappended bibliography.

Tassone et al. (2004) have reported excellent binding of the murine IgG1antibody B-B4 to the CD138 antigen expressed on the surface of MM cells.Tassone also reported high cytotoxic activity of the immunoconjugateB-B4-DM1, which comprises the maytansinoid DM1 as an effector molecule,against multiple myeloma cells (see also US Patent Publ. 20070183971).

Tassone also showed that his B-B4 conjugate was effective even in thebone marrow microenvironment which induces in multiple myeloma (MM)cells resistance to many drugs commonly administered to MM patients,including, for example dexamethasone. Tassone's immunoconjugates wereable to effectively destroy MM tumor cells in the bone marrow stromaenvironment.

While Tassone et al. have contributed to providing an effectivetreatment of MM and a composition of matter that may be employed in sucha treatment, there remain a number of needs in the art.

There remains a need for improved treatments using immunoconjugatesbased on B-B4. There is also a need for effective treatments employingB-B4 based immunoconjugates that show one or more advantageousproperties. Properties of the immunoconjugate preferably includeimproved antigen binding, improved killing of tumor cells comprising, inparticular of CD138 expressing tumor cells, and cells accessory theretoor more homogenous binding of the target, but in particular the abilityto more effectively combat disease states associated with CD138expressing cells.

SUMMARY OF THE INVENTION

The invention is directed at a method for diminishing adhesion of stromacells to CD138 expressing tumor cells in tumor cells of a subject inneed thereof comprising:

administering to said tumor cells an immunoconjugate targeting saidCD138 expressing tumor cells, in particular one containing a cleavablelinker and an effector as disclosed herein, in an amount effective todiminish adhesion of stroma cells to CD138 expressing tumor cells, andoptionally administering to said tumor cells a further cytotoxic agentin a growth of tumor cells inhibiting, delaying and/or preventingamount.

The adhesion may be diminished by at least about 10%, about 20%, about30%, about 40%, about 50%, about 60%, about 70%, about 80% or moreand/or may results in alleviation of adhesion mediated drug resistance,which includes adhesion mediated drug resistance against a furthercytotoxic agent (that is not one of the above immunoconjugates) andwherein said further cytotoxic agent is administered in a growth oftumor cells inhibiting, delaying and/or preventing amount. Theimmunoconjugate and this cytotoxic agent(s) may thus be administeredconsecutively, wherein administration of the cytotoxic agent may followthe administration of the immunoconjugate or may be co-administered.

The immunoconjugate of the present invention in particular comprises

(a) an engineered targeting antibody, and(b) an effector molecule, wherein said immunoconjugate homogenouslytargets CD138 expressing target cells.

The engineered targeting antibody of the present invention may

(i) consist essentially of antigen binding region (ABR) against CD138 ofa non-human antibody, or

(ii) comprise an antigen binding region (ABR) against CD138, whereinsaid antigen binding region is of a non-human antibody, and a furtherantibody region, wherein at least part of said further antibody regionis of a human antibody.

The ABR may comprise:(a) heavy chain variable region CDR3 comprising amino acid residues 99to 111 of SEQ ID NO: 1, and(b) light chain variable region CDR3 comprising amino acid residues 89to 97 of SEQ ID NO: 2, respectively.The ABR may further comprise:(a) heavy chain variable region CDR1 and CDR2 comprising amino acidresidues 31 to 35 and 51 to 68 of SEQ ID NO: 1, and/or (b) light chainvariable region CDR1 and CDR 2 comprising amino acid residues 24 to 34and 50 to 56 of SEQ ID NO: 2, respectively.The further antibody region may comprise:(a) amino acid residues 123 to 448 of SEQ ID NO: 1, and/or(b) amino acid residues 108 to 214 of SEQ ID NO: 2, respectively andmutations thereof that

-   -   (i) maintain or lower the antibody-dependent cytotoxicity and/or        complement-dependent cytotoxicity of the engineered targeting        antibody and/or    -   (ii) stabilize the engineered targeting antibody.

The effector molecule may be attached to said engineered targetingantibody via a linker. The linker may comprise a disulfide bond. Theeffector molecule (e.g., DM4) may provide sterical hindrance between thetargeting antibody and the effector molecule. The effector molecule maybe at least one maytansinoid (e.g., DM1, DM3, or DM4) taxane or aCC1065, or an analog thereof.

The immunoconjugate may bind CD138 with a targeting variation of lessthan 150%, 140%, 130%, 120%, 110%, 100%, 90%, 80%, 70%, 60% or 50%.

The present invention is also directed at an immunoconjugate comprising:

a targeting agent targeting CD138 comprising

an isolated polypeptide comprising an amino acid sequence of animmunoglobulin heavy chain or part thereof, wherein said immunoglobulinheavy chain or part thereof has at least 70% sequence identity with SEQID NO:1. A constant region of said immunoglobulin heavy chain or saidpart thereof may be an IgG4 isotype constant region.

The present invention is also directed at a method of treating MM in asubject, comprising:

providing one of more of the immunoconjugates specified herein, andadministering to said subject said immunoconjugate in an amounteffective to treat multiple myeloma.

The targeting agent of the immunoconjugate may comprise a light chainsequence having at least about 70% sequence identity with SEQ ID NO:2.The targeting agent of the immunoconjugate may also comprise a heavychain sequence having at least about 70% sequence identity with SEQ IDNO:1.

The present invention is also directed at a method for immunoconjugatemediated drug delivery comprising:

providing one or more of the immunoconjugates specified herein, andadministering said immunoconjugate in a therapeutically effectiveamount, wherein said IgG4 isotype alleviates ADCC, complement dependentcytotoxicity and/or Fc-mediated targeting of hepatic FcR.

The present invention is also directed at a method for inhibiting,delaying and/or preventing the growth of tumor cells in a cell culturecomprising

administering to said cell culture a growth of tumor cells inhibiting,delaying and/or preventing effective amount of one or more of theimmunoconjugates specified herein. The effective amount may induce celldeath or continuous cell cycle arrest in CD138 expressing tumor cellsand, optionally, auxiliary cells that do not express CD138, inparticular tumor stroma cells. The cells in said cell culture may beobtained from a cancer patient and, after administration of saideffective amount of said immunoconjugate, the cells of said cell culturemay be reimplanted into said cancer patient.

The present invention is also directed at a method for inhibiting,delaying and/or preventing the growth of a tumor comprising CD138 tumorcells and/or spread of tumor cells of such a tumor in a patient in needthereof, comprising

-   -   administering to said patient at least one or more of the        immunoconjugates specified above in a growth of said tumor        and/or spreading of said tumor cells inhibiting or reducing        amount,    -   wherein said immunoconjugate inhibits, delays or prevents the        growth and/or spread of said tumor cells.

The effector molecule of said immunoconjugate(s) may be a toxin,cytotoxic enzyme, low molecular weight cytotoxic drug, a pore-formingagent, biological response modifier, prodrug activating enzyme, anantibody, cytokine or a radionuclide.

Immunoconjugates of the present invention may be administered in asingle dose of 5 mg/m² to about 300 mg/m², optionally at hourly, daily,weekly intervals or combinations thereof.

Multiple dose regimes include, hourly, daily and weekly regimes are partof the present invention and include in particular administration atintervals of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23 hours, 1, 2, 3, 4, 5, 6, 7 days, 1, 2, 3, 4, 5, 6, 7 or 8weeks.

The present invention is also directed at a method for inhibiting,delaying and/or preventing the growth of a tumor and/or spread ofmalignant tumor cells in a patient in need thereof, comprising

-   -   (a) administering to said patient one or more cytotoxic agents        and/or radiation in an amount effective to reduce tumor load;        and    -   (b) administering to said patient at least one of the        immunoconjugates specified herein in a growth of a tumor and/or        spreading of tumor cells inhibiting, delaying or preventing        amount,    -   wherein said immunoconjugate inhibits, delays or prevents the        growth and/or spread of tumor cells comprising CD138 expressing        cells.

The cytotoxic agent of any of the embodiments of the present inventionmay, in particular, be mephalan, vincristine, doxorubicin,dexamethasone, cyclophosphamide, etoposide, cytarabine, cisplatin,thalidomide, prednisone, thalidomide, bortezomib, lenalidomide,sorafenib, romidepsin or combinations thereof or may be antibody based.

The present invention is also directed at a method for treating asubject having a condition that would benefit from the suppression ofmyeloma cell survival, the method comprising:

(a) providing at least one of any of the immunoconjugates specifiedherein, and(b) administering the immunoconjugate to the subject to selectivelydecrease survival or growth of said myeloma cells of said subject.

The present invention is also directed at a pharmaceutical compositioncomprising any of the immunoconjugates specified herein for theinhibition, delay and/or prevention of the growth of tumors and/orspread of tumor cells, and one or more pharmaceutically acceptableexcipients.

The pharmaceutical composition may include cytotoxic agents as specifiedherein.

The present invention is also directed at a kit comprising, in separatecontainers, pharmaceutical compositions for use in combination toinhibit, delay and/or prevent the growth of tumors and/or spread oftumor cells, wherein one container comprises an effective amount of theabove pharmaceutical composition, and wherein, a separate containercomprises a second pharmaceutical composition comprising an effectiveamount of an agent, preferably a cytotoxic agent, for the inhibition,delay and/or prevention of the growth of tumors and/or spread of tumorcells, and one or more pharmaceutically acceptable excipients.

The present invention is also directed at a method for inhibiting,delaying and/or preventing growth of a tumor comprising CD138 tumorcells and/or spread of tumor cells of such a tumor in a subject in needthereof, comprising

(a) providing an immunoconjugate comprising:an engineered targeting antibody against CD138 attached to an effectormolecule via a cleavable linker, wherein said effector molecule issterically hindered, and(b) administering to said subject the immunoconjugate of (a) in a growthof said tumor and/or spreading of said tumor cells inhibiting, delayingand/or preventing amount, wherein said immunoconjugate of (a) provides agrowth of a tumor inhibiting activity that exceeds that of itsunhindered counterpart by about 10%, about 20%, about 30%, about 40% ormore.

A growth of a tumor inhibiting activity of an unhindered counterpartcomprising a non-cleavable linker may exceed that of the growth of atumor inhibiting activity of its unhindered counterpart comprising acleavable linker, such as by at least about 5%, at least about 10%, upto about 15%.

Said engineered targeting antibody against CD138 may consist essentiallyof antigen binding region against CD138 of a non-human antibody or maycomprise an antigen binding region against CD138 of a non-human antibodyand a further antibody region, wherein at least part of said furtherantibody region is of a human antibody.

Said cleavable linker may comprise a disulfide bond. The effectormolecule may be DM4. The immunoconjugate may be part of a pharmaceuticalcomposition and may be administered to the subject in at least one dosein an amount from about 5 mg/m² to about 300 mg/m².

The present invention provides an immunoconjugate for use as amedicament wherein the immunoconjugate comprises:(a) an engineered targeting antibody

-   -   (i) consisting essentially of antigen binding region against        CD138 of a non-human antibody, or    -   (ii) comprising an antigen binding region against CD138, wherein        said antigen binding region is of a non-human antibody,        a further antibody region, wherein at least part of said further        antibody region is of a human antibody, and        (b) an effector molecule,        wherein said immunoconjugate homogenously binds to CD138.        The present invention provides a further immunoconjugate for use        as a medicament comprising:

a targeting agent targeting CD138 comprising

an isolated polypeptide comprising an amino acid sequence of animmunoglobulin heavy chain or part thereof, wherein said immunoglobulinheavy chain or part thereof has at least 70% sequence identity with SEQID NO:1.

In particular, in one aspect of the invention the immunoconjugate of theabove paragraph is for use in the treatment of multiple myeloma. Inparticular, the immunoconjugate can be used for the manufacture of amedicament for the treatment of multiple myeloma.

The present invention further provides an immunoconjugate for use inimmunoconjugate mediated drug delivery to a patient, in particular foralleviation of ADCC, complement dependent cytotoxicity and/orFc-mediated targeting of hepatic FcR, wherein the immunoconjugatecomprises a targeting agent targeting CD138 comprising an isolatedpolypeptide comprising an amino acid sequence of an immunoglobulin heavychain or part thereof, wherein said immunoglobulin heavy chain or partthereof has at least 70% sequence identity with SEQ ID NO:1, and whereina constant region of said immunoglobulin heavy chain or part thereof isan IgG4 isotype constant region.

The present invention also provides tumor cells for use in the treatmentof cancer in a patient wherein the tumor cells have been treated in cellculture with an immunoconjugate comprising:(a) an engineered targeting antibody

-   -   (i) consisting essentially of antigen binding region against        CD138 of a non-human antibody, or    -   (ii) comprising an antigen binding region against CD138, wherein        said antigen binding region is of a non-human antibody, a        further antibody region, wherein at least part of said further        antibody region is of a human antibody, and        (b) an effector molecule, wherein said immunoconjugate        homogenously binds to CD138.

The present invention also provides tumor cells for use in the treatmentof cancer in a patient wherein the tumor cells have been treated in cellculture with an immunoconjugate comprising:

a targeting agent targeting CD138 comprising

an isolated polypeptide comprising an amino acid sequence of animmunoglobulin heavy chain or part thereof, wherein said immunoglobulinheavy chain or part thereof has at least 70% sequence identity with SEQID NO:1.

The present invention provides an immunoconjugate for use in inhibiting,delaying and/or preventing the growth of a tumor comprising CD138 tumorcells and/or spread of tumor cells of such a tumor in a patient, whereinthe immunoconjugate comprises:

(a) an engineered targeting antibody

-   -   (i) consisting essentially of antigen binding region against        CD138 of a non-human antibody, or    -   (ii) comprising an antigen binding region against CD138, wherein        said antigen binding region is of a non-human antibody,        a further antibody region, wherein at least part of said further        antibody region is of a human antibody, and        (b) an effector molecule, wherein said immunoconjugate        homogenously binds to CD138.

Alternatively, the present invention provides an immunoconjugate for usein inhibiting, delaying and/or preventing the growth of a tumorcomprising CD138 tumor cells and/or spread of tumor cells of such atumor in a patient, wherein the immunoconjugate comprises:

a targeting agent targeting CD138 comprising

an isolated polypeptide comprising an amino acid sequence of animmunoglobulin heavy chain or part thereof, wherein said immunoglobulinheavy chain or part thereof has at least 70% sequence identity with SEQID NO:1.

Still further, the present invention provides a medicament comprising animmunoconjugate and one or more cancer drugs as a combined preparationfor simultaneous, separate or sequential use in the treatment of tumorcells comprising CD138 expressing cells, wherein the immunoconjugatecomprises:

(a) an engineered targeting antibody

-   -   (i) consisting essentially of antigen binding region against        CD138 of a non-human antibody, or    -   (ii) comprising an antigen binding region against CD138, wherein        said antigen binding region is of a non-human antibody,        a further antibody region, wherein at least part of said further        antibody region is of a human antibody, and        (b) an effector molecule,        wherein said immunoconjugate homogenously binds to CD138,        and wherein the one or more cancer drugs are capable of reducing        the tumor load.

Alternatively, the present invention provides a medicament comprising animmunoconjugate and one or more cancer drugs as a combined preparationfor simultaneous, separate or sequential use in the treatment of tumorcells comprising CD138 expressing cells, wherein the immunoconjugatecomprises:

a targeting agent targeting CD138 comprising

an isolated polypeptide comprising an amino acid sequence of animmunoglobulin heavy chain or part thereof, wherein said immunoglobulinheavy chain or part thereof has at least 70% sequence identity with SEQID NO:1,

and wherein the one or more cancer drugs are capable of reducing thetumor load.

In a further aspect of the use of the above two paragraphs the combinedpreparation is to be administered to a patient who has been treated withradiation.

In an alternative aspect the present invention provides the use of animmunoconjugate for the manufacture of a medicament for treating tumorcells in a patient comprising CD138 expressing cells, wherein theimmunoconjugate comprises:

(a) an engineered targeting antibody

-   -   (i) consisting essentially of antigen binding region against        CD138 of a non-human antibody, or    -   (ii) comprising an antigen binding region against CD138, wherein        said antigen binding region is of a non-human antibody,        a further antibody region, wherein at least part of said further        antibody region is of a human antibody, and        (b) an effector molecule,        wherein said immunoconjugate homogenously binds to CD138,        and wherein the medicament is to be administered to a patient        treated with radiation to reduce the tumor load.

Still further the present invention provides the use of animmunoconjugate for the manufacture of a medicament for treating tumorcells in a patient comprising CD138 expressing cells, wherein theimmunoconjugate comprises:

a targeting agent targeting CD138 comprising an isolated polypeptidecomprising an amino acid sequence of an immunoglobulin heavy chain orpart thereof, wherein said immunoglobulin heavy chain or part thereofhas at least 70% sequence identity with SEQ ID NO:1,

and wherein the medicament is to be administered to a patient treatedwith radiation to reduce the tumor load.

In the above paragraphs, the medicament is capable of inhibiting,delaying and/or preventing the growth of a tumor and/or spread ofmalignant tumor cells in a patient.

Further the present invention provides an immunoconjugate forsuppression of myeloma cell survival in an individual wherein theimmunoconjugate comprises:

(a) an engineered targeting antibody

-   -   (i) consisting essentially of antigen binding region against        CD138 of a non-human antibody, or    -   (ii) comprising an antigen binding region against CD138, wherein        said antigen binding region is of a non-human antibody,        a further antibody region, wherein at least part of said further        antibody region is of a human antibody, and        (b) an effector molecule,        wherein said immunoconjugate homogenously binds to CD138.

Still further the present invention provides an immunoconjugate forsuppression of myeloma cell survival in an individual wherein theimmunoconjugate comprises:

a targeting agent targeting CD138 comprising an isolated polypeptidecomprising an amino acid sequence of an immunoglobulin heavy chain orpart thereof, wherein said immunoglobulin heavy chain or part thereofhas at least 70% sequence identity with SEQ ID NO:1.

In the above two paragraphs the immunoconjugate is, in particular,capable of selectively decreasing the survival or growth of said myelomacells in the individual.

Further, the present invention provides an immunoconjugate for use ininhibiting, delaying and/or preventing growth of a tumor comprisingCD138 tumor cells and/or spread of tumor cells of such a tumor in asubject wherein the immunoconjugate comprises an engineered targetingantibody against CD138 attached to an effector molecule via a cleavablelinker, wherein said effector molecule is sterically hindered.

In the above paragraph, the immunoconjugate is, in particular, capableof providing a tumor growth inhibiting activity that exceeds that of itsunhindered counterpart by about 10%, about 20%, about 30%, about 40% ormore.

The present invention also provides a CD138 targeting agent for use in amethod for diminishing adhesion of stroma cells to CD138 expressingtumor cells in tumor cells of a subject.

The present invention further provides a medicament comprising a CD138targeting agent and a further agent, such as an targetingimmunoconjugate or a cytostatic agent, as a combined preparation forsimultaneous (co-administration), separate or sequential use in a methodof diminishing adhesion of stroma cells to CD138 expressing tumor cellsin tumor cells in a subject.

In the above paragraph, the combined preparation is, in particular,capable of inhibiting, delaying and/or preventing growth of tumor cellsin a subject.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides a schematic representation of nBT062 having effectormolecules attached.

FIG. 2 is a chemical representation of BT062.

FIG. 3 shows the conversion of ansamitocin P-3 to maytansinol(stereochemistry is omitted for simplicity).

FIG. 4 shows a representative synthesis scheme of DM4.

FIG. 5 is a schematic representation of an antibody conjugation (nBT062to DM4).

FIG. 6 shows an analysis of the binding of nBT062-SPDB-DM4,nBT062-SPP-DM1, nBT062-SMCC-DM1 and nBT062 antibody to OPM-2 cells.Different concentrations of nBT062 and conjugates were given to thecells and mean fluorescence was measured by FACS analysis.

FIG. 7(A)-(D) depict in vitro cytotoxicity of nBT062-DMx conjugatestowards MOLP-8 (CD138⁺) and BJAB (CD138⁻) cells . The cells werecultured in flat bottom plates and incubated with the indicatedconcentrations of immunoconjugates for 5 days. WST reagent was added forfurther 3 hours to assess cell viability. In (D) cytotoxic activity ofnBT062-SPDB-DM4 was analyzed in the presence or absence of blockingantibody (1 μM nBT062).

FIG. 8 shows tumor volumes for individual mice treated with (A) PBS, (B)nBT062 antibody, (C) free DM4 or (D) non-targeting conjugate huC242-DM4over time (days) post-inoculation with MOLP-8 tumor cells.

FIG. 9 shows tumor volumes for individual mice treated with (A) PBS, (B)nBT062-SPDB-DM4, (C) B-B4-SPP-DM1 or (D) nBT062-SPP-DM1 over time (days)post-inoculation with MOLP-8 tumor cells.

FIG. 10 depicts mean tumor volume (+/−SD) of MOLP-8 human multiplemyeloma xenografts in CB.17 SCID mice over time (days) post-inoculation.

FIGS. 11A and B show the anti-tumor activity of nBT062-DMx againstCD138⁺MOLP-8 tumor cells in a bulky MOLP-8 tumor model in SCID mice.Tumor volume is given as mean (+/−SD) for each group.

FIG. 12 is a graph reflecting the anti-tumour efficacy of nBT062containing DMx conjugates in the SCIDhu/INA-6 model towards multiplemyeloma cells in the environment of human bone marrow. Soluble humanIL-6 Receptor produced by multiple myeloma cells (shuIL-6R) was used asan indicator for tumor burden. Triangle: nBT062-SPP-DM1, Square:nBT062-SPDB-DM4; Diamond: vehicle control.

FIG. 13 shows nBT062-SPDB-DM4 mediated bystander killing in vitro. CD138positive OPM2 cells and CD138 negative Namawla cells were cultured withnBT062-SPDB-DM4 at different concentrations and cell viablility wasmeasured. OD₄₅₀ values represent a measure for cell viability.

FIG. 14 shows in (A) CD138 expression by different MM cells and in (B) amicroscopic analysis of DOX40 cells (upper panel) and OPM1 cells (lowerpanel). CD138 expression is shown at the right hand side, nucleic acidsis shown on the left hand side.

FIG. 15 (A) depicts the cytotoxicity of nBT062-SMCC-DM1, nBT062-SPDB-DM4and nBT062-SPP-DM1 towards MM cells after 40, 80 and 120 hours. FIG. 15(B) depicts the cell viability of MM cells isolated from three differentpatients after treatment with nBT062-SPDB-DM4 for 2 days. FIG. 15 (C)shows Peripheral Blood Mononuclear Cells (PBMCs) derived from 3 healthysubjects that were cultured with nBT062-SPDB-DM4 for 72 h before cellviability was determined.

FIG. 16 (A) shows a cell cycle analysis in which OPM1 cells were treatedwith immunoconjugates for 0, 12 or 24 h and cell cycle profiles wereanalyzed by PI staining. In FIG. 16 (B) OPM1 cells were cultured in thepresence or absence of immunoconjugates for 24, 48 or 72 h. Thepercentage of apoptotic cells was assessed by Apo 2.7 antibody stainingand flow-cytometric analysis. In FIG. 16 (C) OPM1 cells were cultured inthe presence of 885 μg/ml nBT062-SPDB-DM4 for the indicated times (leftpanel) or with different concentrations of immunoconjugate (middlepanel). The pan-caspase inhibitor zVAD-fmk blocked nBT062-SPDB-DM4induced casapase -8, -9, and -3 and Poly (ADP-ribose) polymerase (PARP)cleavage in OPM1 cells (right panel).

FIG. 17 shows in (A) to (C) the effect of IL-6, IGF-1 and BMSCs ongrowth and sensitivity of MM cells towards the immunoconjugates. FIG. 17(D) shows experiments using Dexamethasone instead of immunoconjugate.FIG. 17 (E) shows the results of cell adhesion experiment withco-cultures of tumor cells and BMSCs in the presence or absence ofimmunoconjugates.

FIG. 18 (A) depicts an analysis of GFP expression by OPM1^(GFP) cells.FIG. 18 (B) shows the effect of nBT062-SPDB-DM4, nBT062-SPP-DM1 orbuffer control on tumor sizes in groups of SCID injected with 5×10⁶OPM1^(GFP) cells. FIG. 18 (C) nBT062-SPDB-DM4 significantly increasedsurvival (P<0.0023, dashed line, n=5) compared with the control grouptreated with vehicle only (solid line; normal saline, n=5). FIG. 18 (D)demonstrates that nBT062-SPDB-DM4 induces apoptosis in vivo.

DETAILED DESCRIPTION OF VARIOUS AND PREFERRED EMBODIMENTS OF THEINVENTION

The present invention relates to immunoconjugates comprising CD138targeting agents and the delivery of the effector molecule(s) of theimmunoconjugates to target sites and the site specific release ofeffector(s) molecule in, at or near target cells, tissues and organs.The effector molecules may be activated by cleavage/dissociation fromthe targeting agent portion of the immunoconjugate at the target site.The present invention is in particular related to the use of theseimmunoconjugates in combating tumor growth in vivo and to the use of theability of these immunoconjugates to overcome or diminish protectivemechanisms encountered in the bone marrow microenvironment.

The immunoconjugates according to the present invention may beadministered to a subject in need of therapeutic treatment or to cellsisolated from such a subject in need of therapeutic treatment. Theeffector molecule or molecules may be released from the immunoconjugateby cleavage/dissociation in, at or close to the target cell, tissue ororgan.

In one example, the immunoconjugate comprises the antibody nBT062, whichtargets CD138 expressing cells, and at least one highly cytotoxic drugor toxin as an effector molecule, is administered to a patient withcancer. In this example, a therapeutically effective amount of theimmunoconjugate is administered intravenously to a patient so that itconcentrates in the cancer cells. The effector molecule or molecules arethen released from the antibody by natural means. After or duringcleavage the effector molecule may be stabilized by alkylation and maydiffuse to surrounding auxiliary cells such as stroma cells that do notexpress CD138.

In a second example, the immunoconjugate comprises the antibody nBT062,which targets CD138 expressing cells, and at least one highly cytotoxicdrug or toxin as an effector molecule, and an additional cytotoxic agentis administered to a patient with cancer. In this example, atherapeutically effective amount of the immunoconjugate and thecytotoxic agent are administered consecutively. First theimmunoconjugate is administered intravenously to a patient so that itconcentrates in the cancer cells in the bone marrow microenvironment.The immunoconjugate substantially overcomes cell adhesion mediated drugresistance (CAM-DR) and destroys a substantial portion of the CD138expressing tumor cells in the bone marrow microenvironment. Inparticular, the effector molecule(s) are released from the antibody bynatural means and destroy tumor cells. The immunoconjugate at least inpart prevents adhesion of the tumor cells to the stroma cells, some ofwhich may be destroyed by diffusing effector molecule. After a timeinterval of 12 hours, the cytotoxic agent is administered. The cytotoxicagent, whose activity is normally at least reduced by CAM-DR can act ontumor cells that are not associated with stroma cells and destroys CD138expressing tumor cells, which escaped action by the immunoconjugate.

CD138 or syndecan-1 (also described as SYND1; SYNDECAN; SDC; SCD1; CD138ANTIGEN, SwissProt accession number: P18827 human) is a membraneglycoprotein that was originally described to be present on cells ofepithelial origin, and subsequently found on hematopoietic cells(Sanderson, 1989). CD138 has a long extracellular domain that binds tosoluble molecules (e.g., the growth factors EGF, FGF, HGF) and toinsoluble molecules (e.g., to the extracellular matrix componentscollagen and fibronectin) through heparan sulfate chains (Langford,1998; Yang, 2007) and acts as a receptor for the extracellular matrix.CD138 also mediates cell to cell adhesion through heparin-bindingmolecules expressed by adherent cells. It has been shown that CD138 hasa role as a co-receptor for growth factors of myeloma cells (Bisping,2006). Studies of plasma cell differentiation showed that CD138 mustalso be considered as a differentiation antigen (Bataille, 2006).

In malignant hematopoiesis, CD138 is highly expressed on the majority ofMM cells, ovarian carcinoma, kidney carcinoma, gall bladder carcinoma,breast carcinoma, prostate cancer, lung cancer, colon carcinoma cellsand cells of Hodgkin's and non-Hodgkin's lymphomas, chronic lymphocyticleukemia (CLL) (Horvathova, 1995), acute lymphoblastic leukemia (ALL),acute myeloblastic leukemia (AML) (Seftalioglu, 2003 (a); Seftalioglu,2003 (b)), solid tissue sarcomas, colon carcinomas as well as otherhematologic malignancies and solid tumors that express CD138 (Carbone etal., 1999; Sebestyen et al., 1999; Han et al., 2004; Charnaux et al.,2004; O'Connell et al., 2004; Orosz and Kopper, 2001).

Other cancers that have been shown to be positive for CD138 expressionare many ovarian adenocarcinomas, transitional cell bladder carcinomas,kidney clear cell carcinomas, squamous cell lung carcinomas; breastcarcinomas and uterine cancers (see, for example, Davies et al., 2004;Barbareschi et al., 2003; Mennerich et al., 2004; Anttonen et al., 2001;Wijdenes, 2002).

In the normal human hematopoietic compartment, CD138 expression isrestricted to plasma cells (Wijdenes, 1996; Chilosi, 1999) and CD138 isnot expressed on peripheral blood lymphocytes, monocytes, granulocytes,and red blood cells. In particular, CD34⁺ stem and progenitor cells donot express CD138 and anti-CD138 mAbs do not affect the number of colonyforming units in hematopoietic stem cell cultures (Wijdenes, 1996). Innon-hematopoietic compartments, CD138 is mainly expressed on simple andstratified epithelia within the lung, liver, skin, kidney and gut. Onlya weak staining was seen on endothelial cells (Bernfield, 1992; Vooijs,1996). It has been reported that CD138 exists in polymorphic forms inhuman lymphoma cells (Gattei, 1999).

Monoclonal antibodies B-B4, BC/B-B4, B-B2, DL-101, 1 D4, MI15, 1.BB.210,2Q1484, 5F7, 104-9, 281-2 in particular B-B4 have been reported to bespecific to CD138. Of those B-B4, 1D4 and MI15 recognized both theintact molecule and the core protein of CD138 and were shown torecognize either the same or closely related epitopes (Gattei, 1999).Previous studies reported that B-B4 did not recognize soluble CD138, butonly CD138 in membrane bound form (Wijdenes, 2002).

B-B4, a murine IgG1 mAb, binds to a linear epitope between residues90-95 of the core protein on human syndecan-1 (CD138) (Wijdenes, 1996;Dore, 1998). Consistent with the expression pattern of CD138, B-B4 wasshown to strongly react with plasma cell line RPMI8226, but not to reactwith endothelial cells. Also consistent with the expression pattern ofCD138, B-B4 also reacted with epithelial cells lines A431 (keratinocytederived) and HepG2 (hepatocyte derived). An immunotoxin B-B4-saporin wasalso highly toxic towards the plasma cell line RPMI8226, in factconsiderably more toxic than free saporin. However, from the twoepithelial cell lines tested, B-B4-saporin showed only toxicity towardscell line A431, although in a clonogenic assay B-B4 saporin showed noinhibitory effect on the outgrowth of A431 cells (Vooijs, 1996). Otherresearchers reported lack of specificity of MM-associated antigensagainst tumors (Couturier, 1999).

An antibody/immunoconjugate “consisting essentially of” certaincomponents means in the context of the present invention that theantibody/immunoconjugate consists of the specified components and anyadditional materials or components that do not materially affect thebasic characteristics of the antibody.

The present invention uses the term “tumor cell” to include cancer cellsas well as pre-cancerous cells which may or may not form part of a solidtumor.

A “targeting agent” according to the present invention is able toassociate with a molecule expressed by a target cell and includespeptides and non-peptides. In particular, targeting agents according tothe present invention include targeting antibodies andnon-immunoglobulin targeting molecules, which may be based onnon-immunoglobulin proteins, including, but not limited to, AFFILIN®molecules, ANTICALINS® and AFFIBODIES®. Non-immunoglobulin targetingmolecules also include non-peptidic targeting molecules such astargeting DNA and RNA oligonucleotides (aptamers), but alsophysiological ligands, in particular ligands of the antigen in question,such as CD138.

A “targeting antibody” according to the present invention is or is basedon a natural antibody or is produced synthetically or by geneticengineering and binds to an antigen on a cell or cells (target cell(s))of interest. A targeting antibody according to the present inventionincludes a monoclonal antibody, a polyclonal antibody, a multispecificantibody (for example, a bispecific antibody), or an antibody fragment.The targeting antibody may be engineered to, for example, improve itsaffinity to the target cells (Ross, 2003) or diminish itsimmunogenicity. The targeting antibody may be attached to a liposomalformulation including effector molecules (Carter, 2003). An antibodyfragment comprises a portion of an intact antibody, preferably theantigen binding or variable region of the intact antibody. Examples ofantibody fragments according to the present invention include Fab, Fab′,F(ab′)₂, and Fv fragments, but also diabodies; domain antibodies (dAb)(Ward, 1989; U.S. Pat. No. 6,005,079); linear antibodies; single-chainantibody molecules; and multispecific antibodies formed from antibodyfragments. In a single chain variable fragment antibody (scFv) the heavyand light chains (VH and VL) can be linked by a short amino acid linkerhaving, for example, the sequence (glycine₄serine)_(n), which hassufficient flexibility to allow the two domains to assemble a functionalantigen binding pocket. Addition of various signal sequences may allowfor more precise targeting of the targeting antibody. Addition of thelight chain constant region (CL) may allow dimerization via disulphidebonds, giving increased stability and avidity. Variable regions forconstructing the scFv can, if a mAb against a target of interest isavailable, be obtained by RT-PCR which clones out the variable regionsfrom mRNA extracted from the parent hybridoma. Alternatively, the scFvcan be generated de novo by phage display technology (Smith, 2001). Asused herein, the term “functional fragment”, when used in reference to atargeting antibody, is intended to refer to a portion of the targetingantibody which is capable of specifically binding an antigen that isspecifically bound by the antibody reference is made to. A bispecificantibody according to the present invention may, for example, have atleast one arm that is reactive against a target tissue and one arm thatis reactive against a linker moiety (United States Patent Publication20020006379). A bispecific antibody according to the present inventionmay also bind to more than one antigen on a target cell (Carter, 2003).An antibody according to the present invention may be modified by, forexample, introducing cystein residues to introduce thiol groups(Olafsen, 2004).

In accordance with the present invention, the targeting antibody may bederived from any source and may be, but is not limited to, a camelantibody, a murine antibody, a chimeric human/mouse antibody or achimeric human/monkey antibody, in particular, a chimeric human/mouseantibody such as nBT062.

Humanized antibodies are antibodies that contain sequences derived froma human-antibody and from a non-human antibody and are also within thescope of the present invention. Suitable methods for humanizingantibodies include CDR-grafting (complementarity determining regiongrafting) (EP 0 239 400; WO 91/09967; U.S. Pat. Nos. 5,530,101; and5,585,089), veneering or resurfacing (EP 0 592 106; EP 0 519 596;Padlan, 199; Studnicka et al., 1994; Roguska et al., 1994), chainshuffling (U.S. Pat. No. 5,565,332) and Delmmunosation™ (Biovation,LTD). In CDR-grafting, the mouse complementarity-determining regions(CDRs) from, for example, mAb B-B4 are grafted into human variableframeworks, which are then joined to human constant regions, to create ahuman B-B4 antibody (hB-B4). Several antibodies humanized byCDR-grafting are now in clinical use, including MYLOTARG (Sievers etal., 2001) and HECEPTIN (Pegram et al, 1998).

The resurfacing technology uses a combination of molecular modeling,statistical analysis and mutagenesis to alter the non-CDR surfaces ofantibody variable regions to resemble the surfaces of known antibodiesof the target host. Strategies and methods for the resurfacing ofantibodies, and other methods for reducing immunogenicity of antibodieswithin a different host, are disclosed, for example, in U.S. Pat. No.5,639,641. Human antibodies can be made by a variety of methods known inthe art including phage display methods. See also U.S. Pat. Nos.4,444,887, 4,716,111, 5,545,806, and 5,814,318; and international patentapplication publications WO 98/46645, WO 98/50433, WO 98/24893, WO98/16654, WO 96/34096, WO 96/33735, and WO 91/10741.

Targeting antibodies that have undergone any non-natural modificationsuch as chimeric human/mouse antibodies or a chimeric human/monkeyantibodies, humanized antibodies or antibodies that were engineered to,for example, improve their affinity to the target cells or diminishtheir immunogenicity but also antibody fragments, in particularfunctional fragments of such targeting antibodies that have undergoneany non-natural modification, diabodies; domain antibodies; linearantibodies; single-chain antibody molecules; and multispecificantibodies are referred to herein as engineered targeting antibodies.

Chimerized antibodies, maintain the antibody binding region (ABR or Fabregion) of the non-human antibody, e.g., the murine antibody they arebased on, while any constant regions may be provided for by, e.g., ahuman antibody. Generally, chimerization and/or the exchange of constantregions of an antibody will not affect the affinity of an antibodybecause the regions of the antibody which contribute to antigen bindingare not affected by this exchange. In a preferred embodiment of thepresent invention, the engineered, in particular chimerized, antibody ofthe present invention, may have a higher binding affinity (as expressedby K_(D) values) than the respective non-human antibody it is based on.In particular, the nBT062 antibody and antibodies based thereon may havehigher antibody affinity than the murine B-B4. In another preferredembodiment of the present invention, immunoconjugates comprising thoseengineered/chimerized antibodies also display this higher antibodyaffinity. These immunoconjugates may also display in certain embodimentsother advantageous properties, such as a higher reduction of tumor loadthan their B-B4 containing counterparts. In a preferred embodiment, theengineered, in particular chimerized targeting antibodies displaybinding affinities that are characterized by dissociation constantsK_(D) (nM) of less than 1.6, less than 1.5 or about or less than 1.4,while their murine counterparts are characterized by dissociationconstants K_(D) (nM) of about or more than 1.6. Immunoconjugatescomprising targeting agents such as targeting antibodies may becharacterized by dissociation constants of K_(D) (nM) of less than 2.6,less than 2.5, less than 2.4, less than 2.3, less than 2.2, less than2.1, less than 2.0, less than or about 1.9 are preferred, whileimmunoconjugates comprising the murine counterpart antibodies may becharacterized by dissociation constants K_(D) (nM) of about or more than2.6 (compare Table 3, Materials and Methods).

Fully human antibodies may also be used. Those antibodies can beselected by the phage display approach, where CD138 or an antigenicdeterminant thereof is used to selectively bind phage expressing, forexample, B-B4 variable regions (see, Krebs, 2001). This approach isadvantageously coupled with an affinity maturation technique to improvethe affinity of the antibody. All antibodies referred to herein areisolated antibodies.

In one embodiment, the targeting antibody is, in its unconjugated form,moderately or poorly internalized. Moderate internalization constitutesabout 30% to about 75% internalization of antibody, poor internalizationconstitutes about 0.01% to up to about 30% internalization after 3 hoursincubation at 37° C. In another preferred embodiment the targetingantibody binds to CD138, for example, antibodies B-B4, BC/B-B4, B-B2,DL-101, 1 D4, MI15, 1.BB.210, 2Q1484, 5F7, 104-9, 281-2 in particularB-B4. Hybridoma cells, which were generated by hybridizing SP02/0myeloma cells with spleen cells of Balb/c mice have been deposited withthe DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH,Mascheroder Weg 1, D-38124 Braunschweig on Dec. 11, 2007. Theidentification number of these B-B4 expressing hybridoma cells is DSMACC2874. In another embodiment, the targeting antibody does notsubstantially bind non-cell-surface expressed CD138. When, in thecontext of the present invention, the name of a specific antibody iscombined with the term “targeting antibody” such as “nBT062 targetingantibody,” this means that this targeting antibody has the bindingspecificity of the antibody nBT062. If a targeting antibody is said tobe “based on” a specified antibody, this means that this targetingantibody has the binding specificity of this antibody, but might takeany form consistent with the above description of a targeting antibody.When, in the context of the present invention, the name of a specificantigen is combined with the term “targeting antibody” such as “CD138targeting antibody,” this means that this targeting antibody has thebinding specificity for CD138. If, in the context of the presentinvention, for example, a targeting antibody is said to do something“selectively” such as “selectively targeting cell-surface expressedCD138” or, to be “selective” for something, this means that there is asignificant selectivity (i.e. a higher affinity towards CD138-positivecells compared with CD138-negative cells) for, in the case of theexample provided, cell-surface expressed CD138, compared to any otherantigens. Adverse side effects in a given environment are substantiallyreduced or even avoided due to this selectivity.

“Non-immunoglobulin targeting molecules” according to the presentinvention include targeting molecules derived from non-immunoglobulinproteins as well as non-peptidic targeting molecules. Smallnon-immunoglobulin proteins which are included in this definition aredesigned to have specific affinities towards, in particular surfaceexpressed CD138. These small non-immunoglobulin proteins includescaffold based engineered molecules such as Affilin® molecules that havea relatively low molecular weight such as between 10 kDa and 20 kDa.Appropriate scaffolds include, for example, gamma crystalline. Thosemolecules have, in their natural state, no specific binding activitytowards the target molecules. By engineering the protein surfacesthrough locally defined randomization of solvent exposed amino acids,completely new binding sites are created. Former non-binding proteinsare thereby transformed into specific binding proteins. Such moleculescan be specifically designed to bind a target, such as CD138, and allowfor specific delivery of one or more effector molecules (see, scilProteins GmbH at www.scilproteins.com, 2004). Another kind ofnon-immunoglobulin targeting molecules are derived from lipocalins, andinclude, for example ANTICALINS®, which resemble in structure somewhatimmunoglobulins. However, lipocalins are composed of a singlepolypeptide chain with 160 to 180 amino acid residues. The bindingpocket of lipocalins can be reshaped to recognize a molecule of interestwith high affinity and specificity (see, for example, Beste et al.,1999). Artificial bacterial receptors such as those marketed under thetrademark Affibody® (Affibody AB) are also within the scope of thepresent invention. These artificial bacterial receptor molecules aresmall, simple proteins and may be composed of a three-helix bundle basedon the scaffold of one of the IgG-binding domains of Protein A(Staphylococcus aureus). These molecules have binding properties similarto many immunoglobulins, but are substantially smaller, having amolecular weight often not exceeding 10 kDa and are also comparativelystable. Suitable artificial bacterial receptor molecules are, forexample, described in U.S. Pat. Nos. 5,831,012; 6,534,628 and 6,740,734.

Other “non-immunoglobulin targeting molecules” are physiological ligandsof the antigen in question. Physiological ligands of CD138 include forexample, but not limited to, ADAMTS4 (aggrecanase-1), antithrombin-3,bFGF, cathepsin G, CCL5 (RANTES), CCL7, CCL11, CCL17, CD44, collagens(collagen type 1, collagen type 2, collagen type 3, collagen type 4,collagen type 5, collagen type 6), CXCL1, elastase, gp120, HGF[hepatocyte growth factor], laminin-1, laminin-2, laminin-5, midkine,MMP-7, neutrophil elastase, and pleiotrophin (HBNF, HBGF-8).Non-peptidic targeting molecules include, but are not limited to, to DNAand RNA oligonucleotides that bind to CD138 (aptamers).

An “effector molecule” according to the present invention is a moleculeor a derivative, or an analogue thereof that is attached to a targetingagent, in particular a targeting antibody and/or an engineered targetingantibody, and that exerts a desired effect, for example, apoptosis, oranother type of cell death, or a continuous cell cycle arrest on thetarget cell or cells. Effector molecules according to the presentinvention include molecules that can exert desired effects in a targetcell and include, but are not limited to, toxins, drugs, in particularlow molecular weight cytotoxic drugs, radionuclides, biological responsemodifiers, pore-forming agents, ribonucleases, proteins of apoptoticsignaling cascades with apoptosis-inducing activities, cytotoxicenzymes, prodrug activating enzymes, antisense oligonucleotides,antibodies or cytokines as well as functional derivatives oranalogues/fragments thereof. Toxins may include bacterial toxins, suchas, but not limited to, Diphtheria toxin or Exotoxin A, plant toxins,such as but not limited to, Ricin. Proteins of apoptotic signalingcascades with apoptosis-inducing activities, include, but are notlimited to, Granzyme B, Granzyme A, Caspase-3, Caspase-7, Caspase-8,Caspase-9, truncated Bid (tBid), Bax and Bak.

In a preferred embodiment, the effector increases internal effectordelivery of the immunoconjugate, in particular when the natural form ofthe antibody on which the targeting antibody of the immunoconjugate isbased is poorly internalizable. In another preferred embodiment theeffector is, in its native form, non-selective. In certain embodimentsthe effector has high non-selective toxicity, including systemictoxicity, when in its native form. The “native form” of an effectormolecule of the present invention is an effector molecule before beingattached to the targeting agent to form an immunoconjugate. In anotherpreferred embodiment, the non-selective toxicity of the effectormolecule is substantially eliminated upon conjugation to the targetingagent. In another preferred embodiment, the effector molecule causes,upon reaching the target cell, death or continuous cell cycle arrest inthe target cell. A drug-effector molecule according to the presentinvention includes, but is not limited to, a drug including, forexample, small highly cytotoxic drugs that act as inhibitors of tubulinpolymerization such as maytansinoids, dolastatins, auristatin andcrytophycin; DNA alkylating agents like CC-1065 analogues or derivatives(U.S. Pat. Nos. 5,475,092; 5,585,499; 6,716,821) and duocarmycin;enediyne antibiotics such as calicheamicin and esperamicin; and potenttaxoid (taxane) drugs (Payne, 2003). Maytansinoids and calicheamicinsare particularly preferred. An effector maytansinoid includesmaytansinoids of any origin, including, but not limited to syntheticmaytansinol and maytansinol analogue and derivative. Doxorubicin,daunomycin, methotrexate, vinblastine, neocarzinostatin, macromycin,trenimon and a-amanitin are some other effector molecules within thescope of the present invention. Also within the scope of the presentinvention are antisense DNA molecules as effector molecules. When thename of, for example, a specific drug or class of drugs is combinedherein with the term “effector” or “effector molecule,” reference ismade to an effector of an immunoconjugate according to the presentinvention that is based on the specified drug or class of drugs.

Maytansine is a natural product originally derived from the Ethiopianshrub Maytenus serrata (Remillard, 1975; U.S. Pat. No. 3,896,111). Thisdrug inhibits tubulin polymerization, resulting in mitotic block andcell death (Remillard, 1975; Bhattacharyya, 1977; Kupchan, 1978). Thecytotoxicity of maytansine is 200-1000-fold higher than that ofanti-cancer drugs in clinical use that affect tubulin polymerization,such as Vinca alkaloids or taxol. However, clinical trials of maytansineindicated that it lacked a therapeutic window due to its high systemictoxicity. Maytansine and maytansinoids are highly cytotoxic but theirclinical use in cancer therapy has been greatly limited by their severesystemic side-effects primarily attributed to their poor selectivity fortumors. Clinical trials with maytansine showed serious adverse effectson the central nervous system and gastrointestinal system.

Maytansinoids have also been isolated from other plants including seedtissue of Trewia nudiflora (U.S. Pat. No. 4,418,064)

Certain microbes also produce maytansinoids, such as maytansinol and C-3maytansinol esters (U.S. Pat. No. 4,151,042).

The present invention is directed to maytansinoids of any origin,including synthetic maytansinol and maytansinol analogues which aredisclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870;4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268;4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348;4,331,598; 4,361,650; 4,362,663; 4,364,866; 4,371,533; 4,424,219 and4,151,042.

In a preferred embodiment, the maytansinoid is a thiol-containingmaytansinoid and is more preferably produced according to the processesdisclosed in U.S. Pat. No. 6,333,410 to Chari et al or in Chari et al.(Chari, 1992).

DM-1 (N²-deacetyl-N²-(3-mercapto-1-oxopropyl)-maytansine) is a preferredeffector molecule in the context of the present invention. DM1 is 3- to10-fold more cytotoxic than maytansine, and has been converted into apro-drug by linking it via disulfide bond(s) to a monoclonal antibodydirected towards a tumor-associated antigen. Certain of these conjugates(sometimes called “tumor activated prodrugs” (TAPs)) are not cytotoxicin the blood compartment, since they are activated upon associating witha target cells and internalized, thereby releasing the drug (Blättler,2001). Several antibody-DM1 conjugates have been developed (Payne,2003), and been evaluated in clinical trials. For example, huC242-DM1treatment in colorectal cancer patients was well tolerated, did notinduce any detectable immune response, and had a long circulation time(Tolcher, 2003).

Other particularly preferred maytansinoids comprise a side chain thatcontains a sterically hindered thiol bond such as, but not limited to,maytansinoidsN^(2′)-deacetyl-N^(2′)-(4-mercapto-1-oxopentyl)-maytansine, alsoreferred to as “DM3,” andN^(2′)-deacetyl-N^(2′)-(4-methyl-4-mercapto-1-oxopentyl)-maytansine,also referred to as “DM4.” The synthesis of DM4 is shown in FIGS. 3 and4 and is described elsewhere herein. DM4 differs from DM1 and DM3 inthat it bears methyl groups at its aC. This results in a stericalhindrance when DM4 is attached via a linker in particular, but notlimited to, a linker comprising a disulfide bond, to a targeting agentsuch as nBT062. A wide variety of maytansinoids bearing a stericallyhindered thiol group (possessing one or two substituents, in particularalkyls substituents, such as the methyl substituents of DM4) aredisclosed U.S. Patent Publication 2004/0235840, published Nov. 25, 2004,which is incorporated herein in its entirety by reference. The sterichindrance conferred by alkyl groups such as the methyl groups on thecarbon adjacent to the sulfur atom of DM3 and DM4 may affect the rate ofintracellular cleavage of the immunoconjugate. The variable alkyl unitmay therefore affect potency, efficacy, and safety/toxicity in vitro andin vivo.

As reported by Goldmahker et al. in U.S. Patent Publication2006/0233814, such a hindrance induces alkylation (e.g., methylation) ofthe free drug, once the drug is released at its target. The alkylationmay increase the stability of the drug allowing for the so-calledbystander effect. However, as the person skilled in the art willappreciate, other effector molecules comprising substitutents such asalkyl groups at positions that result in a sterical hindrance when theeffector is attached to a targeting agent via a linker are part of thepresent invention (U.S. Patent Publication 2004/0235840). Preferablythis hindrance induces a chemical modification such as alkylation of thefree drug to increase its overall stability, which allows the drug tonot only induce cell death or continuous cell cycle arrest in CD138expressing tumor cells but, optionally, also to affect auxiliary cellsthat, e.g., support or protect the tumor from drugs, in particular cellsof the tumor stroma and the tumor vasculature and which generally do notexpress CD138 to diminish or lose their supporting or protectingfunction.

DNA alkylating agents are also particularly preferred as effectormolecules and include, but are not limited to, CC-1065 analogues orderivatives. CC-1065 is a potent antitumor-antibiotic isolated fromcultures of Streptomyces zelensis and has been shown to be exceptionallycytotoxic in vitro (U.S. Pat. No. 4,169,888). Within the scope of thepresent invention are, for example the CC-1065 analogues or derivativesdescribed in U.S. Pat. Nos. 5,475,092, 5,585,499 and 5,739,350. As theperson skilled in the art will readily appreciate, modified CC-1065analogues or derivatives as described in U.S. Pat. No. 5,846,545 andprodrugs of CC-1065 analogues or derivatives as described, for example,in U.S. Pat. No. 6,756,397 are also within the scope of the presentinvention. In certain embodiments of the invention, CC-1065 analogues orderivatives may, for example, be synthesized as described in U.S. Pat.No. 6,534,660.

Another group of compounds that make preferred effector molecules aretaxanes, especially highly potent ones and those that contain thiol ordisulfide groups. Taxanes are mitotic spindle poisons that inhibit thedepolymerization of tubulin, resulting in an increase in the rate ofmicrotubule assembly and cell death. Taxanes that are within the scopeof the present invention are, for example, disclosed in U.S. Pat. Nos.6,436,931; 6,340,701; 6,706,708 and United States Patent Publications20040087649; 20040024049 and 20030004210. Other taxanes are disclosed,for example, in U.S. Pat. No. 6,002,023, U.S. Pat. No. 5,998,656, U.S.Pat. No. 5,892,063, U.S. Pat. No. 5,763,477, U.S. Pat. No. 5,705,508,U.S. Pat. No. 5,703,247 and U.S. Pat. No. 5,367,086. As the personskilled in the art will appreciate, PEGylated taxanes such as the onesdescribed in U.S. Pat. No. 6,596,757 are also within the scope of thepresent invention.

Calicheamicin effector molecules according to the present inventioninclude gamma 1l, N-acetyl calicheamicin and other derivatives ofcalicheamicin. Calicheamicin binds in a sequence-specific manner to theminor groove of DNA, undergoes rearrangement and exposes free radicals,leading to breakage of double-stranded DNA, resulting in cell apoptosisand death. One example of a calicheamicin effector molecule that can beused in the context of the present invention is described in U.S. Pat.No. 5,053,394.

An immunoconjugate according to the present invention comprises at leastone targeting agent, in particular targeting antibody and one effectormolecule. The immunoconjugate might comprise further molecules forexample for stabilization. For immunoconjugates, the term “conjugate” isgenerally used to define the operative association of the targetingagent with one or more effector molecules and is not intended to refersolely to any type of operative association, and is particularly notlimited to chemical “conjugation”. So long as the targeting agent isable to bind to the target site and the attached effector functionssufficiently as intended, particularly when delivered to the targetsite, any mode of attachment will be suitable. The conjugation methodsaccording to the present invention include, but are not limited to,direct attachment of the effector molecule to the targeting antibody,with or without prior modification of the effector molecule and/or thetargeting antibody or attachment via linkers. Linkers can be categorizedfunctionally into, for example, acid labile, photolabile linkers, enzymecleavable linkers, such as linkers that can be cleaved by peptidases.Cleavable linkers are, in many embodiments of the invention preferred.Such cleavable linkers can be cleaved under conditions present in thecellular environment, in particular, an intracellular environment andthat have no detrimental effect on the drug released upon cleavage. LowpHs such as pH of 4 to 5 as they exist in certain intracellulardepartments will cleave acid labile linkers, while photolabile linkerscan be cleaved by, e.g., infrared light. However, linkers that arecleaved by/under physiological conditions present in the majority ofcells are preferred and are referred to herein as physiologicallycleavable linkers. Accordingly, disulfide linkers are being preferred inmany embodiments of the invention. These linkers are cleavable throughdisulfide exchange, which can occur under physiological conditions.Preferred heterobifunctional disulfide linkers include, but are notlimited to, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) (see,e.g., Carlsson et al. (1978)), N-succinimidyl4-(2-pyridyldithio)butanoate (SPDB) (see, e.g., U.S. Pat. No.4,563,304), N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP) (see,e.g., CAS Registry number 341498-08-6), N-succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (see, e.g.,Yoshitake et al., (1979)), and N-succinimidyl4-methyl-4-[2-(5-nitro-pyridyl)-dithio]pentanoate (SMNP) (see, e.g.,U.S. Pat. No. 4,563,304). The most preferred linker molecules for use inthe inventive composition are SPP, SMCC, and SPDB.

Other suitable linkers may include “non-cleavable” bonds, such as, butnot limited to Sulfosuccinimidyl maleimidomethyl cyclohexane carboxylate(SMCC), which is a heterobifunctional linker capable of linkingcompounds with SH-containing compounds. Bifunctional andheterobifunctional linker molecules, such as carbohydrate-directedheterobifunctional linker molecules, such asS-(2-thiopyridyl)-L-cysteine hydrazide (TPCH), are also within the scopeof the present invention (Vogel, 2004). The effector molecule, such as amaytansinoid, may be conjugated to the targeting antibody via a tworeaction step process, including as a first step modification of thetargeting antibody with a cross-linking reagent such as N-succinimidylpyridyldithiopropionate (SPDP) to introduce dithiopyridyl groups intothe targeting antibody. In a second step, a reactive maytansinoid havinga thiol group, such as DM1, may be added to the modified antibody,resulting in the displacement of the thiopyridyl groups in the modifiedantibody, and the production of disulfide-linked cytotoxicmaytansinoid/antibody conjugate (U.S. Pat. No. 5,208,020). However,one-step conjugation processes such as the one disclosed in UnitedStates Patent Publication 20030055226 to Chari et al are also within thescope of the present invention. In one embodiment of the presentinvention multiple effector molecules of the same or different kind areattached to a targeting antibody. As discussed elsewhere herein, thenature of the linkers employed may influence bystander killing (Kovtunet al., 2006). See also discussion of FIG. 13.

CC-1065 analogues or derivatives may be conjugated to the targetingagent via for example PEG linking groups as described in U.S. Pat. No.6,716,821.

Calicheamicins may be conjugated to the targeting antibodies via linkers(U.S. Pat. No. 5,877,296 and U.S. Pat. No. 5,773,001) or according tothe conjugation methods disclosed in U.S. Pat. No. 5,712,374 and U.S.Pat. No. 5,714,586. Another preferred method for preparing calicheamicinconjugates is disclosed in Unites States Patent Publication 20040082764.The immunoconjugates of the present invention may take the form ofrecombinant fusion proteins.

The term “cytotoxic agents” comprises “cytotoxic/cancer drugs” includingchemotherapeutic agents such as melphalan, cyclophosphamide,vincristine, doxorubicin and liposomal doxorubicin (DOXIL),cyclophosphamide, etoposide, cytarabine and cisplatin, corticosteroidssuch as prednisone and dexamethasone and agents such as thalidomide,bortezomib, lenalidomide, but also kinase inhibitor such as sorafenib orHDAC (histone deacetylase) inhibitors such as romidepsin as well asgrowth inhibitory agents, anti-hormonal agents, anti-angiogenic agents,cardioprotectants, immunostimulatory agents, immunosuppressive agents,angiogenesis inhibitors, protein tyrosine kinase (PTK) inhibitors. Alsoincluded in this definition are antibody based cytotoxic agentsincluding immunoconjugates and antibodies that have an art recognizedcytotoxic effect. Anti-CD40 is a preferred antibody. Other antibodiesinclude, but are not limited to, e.g., AVASTIN (bevacizuab) orMYELOMACIDE (milatuzumab).

THALOMID (a-(N-phthalimido) glutarimide; thaliomide), is animmunomodulatory agent. The empirical formula for thalidomide isC₁₃H₁₀N₂O₄ and the gram molecular weight is 258.2. The CAS number ofthalidomide is 50-35-1. It appears to have multiple actions, includingthe ability to inhibit the growth and survival of myeloma cells invarious ways and to inhibit the growth of new blood vessels.

VELCADE is a proteasome inhibitor used to treat multiple myeloma. It isbelieved that VELCADE acts on myeloma cells to cause cell death, and/oracts indirectly to inhibit myeloma cell growth and survival by acting onthe bone microenvironment. Without being limited to a specific theory ormode of action, VELCADE thus disrupts normal cellular processes,resulting in proteasome inhibition that promotes apoptosis.

REVLIMID is an immunomodulatory agent. It is thought that REVLIMIDaffects multiple pathways in myeloma cells, thereby inducing apoptosis,inhibiting myeloma cell growth, inhibiting vascular endothelial growthfactor (VEGF) thereby inhibiting angiogenesis, and reducing adhesion ofmyeloma cells to bone marrow stromal cells.

Dexamethasone is a synthetic glucocorticoid steroid hormone that acts asan anti-inflammatory and immunosuppressant. When administered to cancerpatients, dexamethasone can counteract side effects of cancer therapy.Dexamethasone can also be given alone or together with other anticanceragents, including thalidomide, adriamycin or vincristine.

The term “in combination with” is not limited to the administration atexactly the same time. Instead, the term encompassed administration ofthe immunoconjugate of the present invention and the other regime (e.g.radiotherapy) or agent, in particular the cytotoxic agents referred toabove in a sequence and within a time interval such that they may acttogether to provide a benefit that is increased compared to treatmentwith only either the immunoconjugate of the present invention or, e.g.,the other agent or agents. It is preferred that the immunoconjugate andthe other agent or agents act additively, and especially preferred thatthey act synergistically. Such molecules are suitably provided inamounts that are effective for the purpose intended. The skilled medicalpractitioner can determine empirically, or by considering thepharmacokinetics and modes of action of the agents, the appropriate doseor doses of each therapeutic agent, as well as the appropriate timingsand methods of administration. As used in the context of the presentinvention “co-administration” refers to administration at the same timeas the immunoconjugate, often in a combined dosage form.

The term “sequence identity” refers to a measure of the identity ofnucleotide sequences or amino acid sequences. In general, the sequencesare aligned so that the highest order match is obtained. “Identity”, perse, has recognized meaning in the art and can be calculated usingpublished techniques. (See, e.g.: Computational Molecular Biology, Lesk,A. M., ed., Oxford University Press, New York, 1988; Biocomputing:Informatics and Genome Projects, Smith, D. W., ed., Academic Press, NewYork, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M.,and Griffin, H. G., eds., Humana Press, New Jersey, 1994; SequenceAnalysis in Molecular Biology, von Heinje, G., Academic Press, 1987; andSequence Analysis Primer, Gribskov, M. and Devereux, J., eds., MStockton Press, New York, 1991). While there exist a number of methodsto measure identity between two polynucleotide or polypeptide sequences,the term “identity” is well known to skilled artisans (Carillo, H. &Lipton, D., SIAM J Applied Math 48:1073 (1988)).

Whether any particular nucleic acid molecule is at least 50%, 60%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, forinstance, the nBT062 nucleic acid sequence, or a part thereof, can bedetermined conventionally using known computer programs such as DNAsissoftware (Hitachi Software, San Bruno, Calif.) for initial sequencealignment followed by ESEE version 3.0 DNA/protein sequence software(cabot@trog.mbb.sfu.ca) for multiple sequence alignments.

Whether the amino acid sequence is at least 50%, 60%, 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance SEQ IDNO:1 or SEQ ID NO:2, or a part thereof, can be determined conventionallyusing known computer programs such the BESTFIT program (WisconsinSequence Analysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, 575 Science Drive, Madison, Wis. 53711).BESTFIT uses the local homology algorithm of Smith and Waterman,Advances in Applied Mathematics 2:482-489 (1981), to find the bestsegment of homology between two sequences.

When using DNAsis, ESEE, BESTFIT or any other sequence alignment programto determine whether a particular sequence is, for instance, 95%identical to a reference sequence according to the present invention,the parameters are set such that the percentage of identity iscalculated over the full length of the reference nucleic acid or aminoacid sequence and that gaps in homology of up to 5% of the total numberof nucleotides in the reference sequence are allowed.

If, in the context of the present invention, reference is made to acertain sequence identity with a combination of residues of a particularsequence, this sequence identity relates to the sum of all the residuesspecified.

The basic antibody molecule is a bifunctional structure wherein thevariable regions bind antigen while the remaining constant regions mayelicit antigen independent responses. The major classes of antibodies,IgA, IgD, IgE, IgG and IgM, are determined by the constant regions.These classes may be further divided into subclasses (isotypes). Forexample, the IgG class has four isotypes, namely, IgG1, IgG2, IgG3, andIgG4 which are determined by the constant regions. Of the various humanantibody classes, only human IgG1, IgG2, IgG3 and IgM are known toeffectively activate the complement system. While the constant regionsdo not form the antigen binding sites, the arrangement of the constantregions and hinge region may confer segmental flexibility on themolecule which allows it to bind with the antigen.

Different IgG isotypes can bind to Fc receptors on cells such asmonocytes, B cells and NK cells, thereby activating the cells to releasecytokines. Different isotypes may also activate complement, resulting inlocal or systemic inflammation. In particular, the different IgGisotypes may bind FcγR to different degrees. FcγRs are a group ofsurface glycoproteins belonging to the Ig superfamily and expressedmostly on leucocytes. The FcγR glycoproteins are divided into threeclasses designated FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16). WhileIgG1, IgG2 and IgG3 bind strongly to a variety of these classes of FcγRglycoproteins, IgG4 display much weaker binding. In particular, IgG4 isan intermediate binder of FcγRI, which results in relatively low or evenno ADCC (antibody dependent cellular cytotoxicity), and does not bind toFcγRIIIA or FcγRIIA. IgG4 is also a weak binder of FcγRIIB, which is aninhibitory receptor. Furthermore, IgG4 mediates only weak or nocomplement fixation and weak or no complement dependent cytotoxicity(CDC). In the context of the present invention, IgG4 may be specificallyemployed to prevent Fc-mediated targeting of hepatic FcR as it displaysno interaction with FcRγII on LSECs (liver sinusoidal endothelialcells), no or weak interaction with FcRγI-III on Kupffer cells(macrophages) and no interaction with FcRγIII on hepatic NK cells.Certain mutations that further reduce any CDC are also part of thepresent invention For example IgG4 residues at positions 327, 330 and331 were shown to reduce ADCC (antibody dependent cellular cytotoxicity)and CDC (Amour, 1999; Shields, 2001). One of more mutations thatstabilize the antibody are also part of the present invention (alsoreferred to herein as “stabilizing mutations”). Those mutations includein particular, leucine-to-glutamic acid mutations in the CH2 region ofIgG4 and serine-to-proline exchanges in the IgG4 hinge core. Thesemutations decrease, in certain embodiments of the invention, the amountof half-molecules to less than 10%, less than 5% and preferably lessthan 2% or 1%. Moreover, the in vivo half life of so stabilizedantibodies might be increased several days, including 1, 2, 3, 4 or morethan 5 days (Schuurman, 1999).

Targeting agents, including targeting antibodies disclosed herein mayalso be described or specified in terms of their binding affinity toantigen, in particular to CD138. Preferred binding affinities oftargeting agents such as targeting antibodies are characterized bydissociation constants K_(D) (nM) of less than 1.6, less than 1.5 orabout or less than 1.4. For immunoconjugates comprising said targetingagents such as targeting antibodies dissociation constants K_(D) (nM) ofless than 1.6, less than 1.5 or less than 2.5, less than 2.4, less than2.3, less than 2.2, less than 2.1, less than 2.0, less than or about 1.9are preferred.

An antigen binding region (ABR) according to the present invention willvary based on the type of targeting antibody or engineered targetingantibody employed. In a naturally occurring antibody and in mostchimeric and humanized antibodies, the antigen binding region is made upof a light chain and the first two domains of a heavy chain. However, ina heavy chain antibody devoid of light chains, the antigen bindingregion will be made up of, e.g., the first two domains of the heavychain only, while in single chain antibodies (ScFv), which combine in asingle polypeptide chain the light and heavy chain variable domains ofan antibody molecule, the ABR is provided by only one polypeptidemolecule. FAB fragments are usually obtained by papain digestion andhave one light chain and part of a heavy chain and thus comprise an ABRwith only one antigen combining site. On the other hand, diabodies aresmall antibody fragments with two antigen-binding regions. In thecontext of the present invention, however, an antigen binding region ofan targeting antibody or engineered targeting antibody is any regionthat primarily determines the binding specificity of the targetingantibody or engineered targeting antibody.If an ABR or another targeting antibody region is said to be “of acertain antibody”, e.g., a human or non-human antibody, this means inthe context of the present invention that the ABR is either identical toa corresponding naturally occurring ABR or is based thereon. An ABR isbased on a naturally occurring ABR if it has the binding specificity ofthe naturally occurring ABR. However, such an ABR may comprise, e.g.,point mutations, additions, deletions or posttranslational modificationsuch as glycosylation. Such an ABR may in particular have more than 70%,more than 80%, more than 90%, preferably more than 95%, more than 98% ormore than 99% sequence identity with the sequence of the naturallyoccurring ABR.Homogenous targeting of a targeting agent such as a targeting antibody,but in particular an immunoconjugate comprising the same, in the contextof the present invention, is a measure of the variance associated withobtaining the desired result of said targeting with the targeting agent.In certain embodiments of the invention, the desired result is obtainedby simple binding to the target. This is, for example, the case inembodiments in which a certain targeting agent provides a shield againstsubsequent binding. However, the homogeneity of a targeting agent can bereadily assessed, e.g., via the efficacy of an immunoconjugatecomprising said targeting agent. For example, the efficacy of saidimmunoconjugate against a tumor antigen such as CD138 that comprises aneffector aimed at destroying tumor cells and/or arresting the growth ofa tumor can be determined by the degree of growth suppression of a tumorcomprising cells expressing the CD138 antigen. Such an immunoconjugatemay display a high variance in its efficacy. It may, for example, arresttumor growth sometimes with high efficacy, but other times with anefficacy that hardly exceeds the efficacy of the control. A low variancein the efficacy of an immunoconjugate, on the other hand, shows that theimmunoconjugate and/or targeting agent, respectively, provide thedesired result consistently. One way of quantifying the homogeneity oftargeting is to calculate the targeting variation. In the context oftumor growth arrested by an immunoconjugate comprising a certaintargeting agent, the targeting variation can be calculated by firstdetermining the time for a tumor to reach a predetermined volume, e.g.300 mm³. Preferably, the predetermined volume is chosen so that anytumor growth before and after reaching said predetermined volume issteadily increasing at about the same rate. After such time has beendetermined for a group of subjects the mean of these times (T_(m)) inthe group of subjects (e.g., SCID mice or another suitable modeldisplaying homogenous tumor growth) is calculated. T_(m) is thencorrelated to the observations made in the subject of the group showingthe least efficacy in targeting and thus being associated with tumorsthat need the least time (T_(f)) to reach said predetermined volume,and, on the other hand, the subject in the group showing the highestefficacy in targeting and thus being associated with tumors that needthe most time (T_(s)) to reach said predetermined volume by calculatingthe targeting variation for the predetermined volume according to thefollowing formula:

TARGETING VARIATION [%]=Ts−Tf/Tm×100

In a preferred embodiment, the targeting variation of theimmunoconjugate comprising the engineered targeting antibody of thepresent invention is less than 150%, less than 140%, less than 130%,less than 120%, less than 110%, less than 100%, less than 90%, less than80%, less than 70%, less than 60%, or less than 50%, and in certainembodiments even less than 45%. Preferably, the targeting variation isbetween about 10% and about 150%, preferably between about 10% and about100%, about 10% and about 80%, about 10% and about 70%, about 10% andabout 60%, about 10% and about 50%.

The homogenity of targeting can be also quantified by other means suchas determining the tumor growth delay. Also, as the person skilled inthe art will readily understand tumor volume of a certain size is onlyone parameter on which basis targeting variation may be determined.Depending on the desired result, other parameters include time (for,e.g., measuring tumor growth delay) or % of binding may be employed. Theperson skilled in the art can readily determine such other parameters.

FIG. 9 shows in (C) and (D) the differences in homogenity oftargeting/binding between immunoconjugates comprising murine antibodyBB4 (BB4-SPP-DM1; FIG. 9C) and the engineered targeting antibody nBT062(nBT062-SPP-DM1; FIG. 9D) based thereon. As can be seen from thesegraphs, results obtained with the immunoconjugate comprising theengineered targeting antibody are substantially more homogenous than theones obtained with the immunoconjugates comprising the murine antibody.This is particularly notable since the antibody binding region of BB4was not modified in nBT062. Thus, the immunoconjugate comprising theantibody binding region of the murine antibody, but no other parts ofthe murine antibody, showed properties that far exceeded results theperson skilled in the art would have expected.

nBT062 (see also FIG. 1) is a murine human chimeric IgG4 mAb achimerized version of B-B4. This chimerized version of B-B4 was createdto reduce the HAMA (Human Anti-Mouse Antibody) response, whilemaintaining the functionality of the antibody binding region of the B-B4for CD138. Surprisingly, the results obtained using an immunoconjugatecomprising the engineered targeting antibody were much more homogenous(the variance in the results was reduced). The protocol for producingnBT062 is specified below. Chinese hamster ovary cells expressing nBT062have been deposited with the DSMZ-Deutsche Sammlung von Mikroorganismenund Zellkulturen GmbH, MascheroderWeg 1, D-38124 Braunschweig on Dec.11, 2007. The identification number is DSM ACC2875. A CD138 specificchimeric antibody based on B-B4 is generically referred to herein asc-B-B4.

The amino acid sequence for both, the heavy and the light chains hasbeen predicted from the translation of the nucleotide sequence fornBT062. The amino acid sequences predicted for the heavy chain and lightchain are presented in Table 1. Predicted variable regions are bolded,predicted CDRs are underlined.

TABLE 1 Predicted Amino Acid Sequence for nBT062 - nBT062 heavy chainpredicted sequence (SEQ ID NO:1): 1 QVQLQQSGSE LMMPGASVKI SCKATGYTFSNYWIE WVKQR PGHGLEWIGE 51 ILPGTGRTIY  NEKFKGKA TF TADISSNTVQ MQLSSLTSEDSAVYYCARRD 101 YYGNFYYAMD  Y WGQGTSVTV SSASTKGPSV FPLAPCSRST SESTAALGCL151 VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT 201KTYTCNVDHK PSNTKVDKRV ESKYGPPCPS CPAPEFLGGP SVFLFPPKPK 251 DTLMISRTPEVTCVVVDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNS 301 TYRVVSVLTV LHQDWLNGKEYKCKVSNKGL PSSIEKTISK AKGQPREPQV 351 YTLPPSQEEM TKNQVSLTCL VKGFYPSDIAVEWESNGQPE NNYKTTPPVL 401 DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVMHEALHNHYTQKSLSLSLG(K) The C-terminal lysine is prone to clipping andmight be present due to incomplete clipping to a certain extent. The (K)in parentesis is not part of SEQ ID NO:1. - nBT062 light chain predictedsequence (SEQ ID NO:2): 1 DIQMTQSTSS LSASLGDRVT ISC SASQGIN   NYLNWYQQKP DGTVELLIYY 51 TSTLQSGVPS RFSGSGSGTD YSLTISNLEP EDIGTYYC QQ  YSKLPRT FGG 101 GTKLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV151 DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG 201LSSPVTKSFN RGEC

Table 2. shows a comparision of the general CDR definitions of Krabatand Chothia and the predicted CDRs for BT062

nBT062 Kabat CDR definition Light chain CDR1: residues 24-34 CDR1:residues 24-34 CDR2: residues 50-56 CDR2: residues 50-56 CDR3: residues89-97 CDR3: residues 89-97 Heavy chain CDR1: residues 31-35 CDR1:residues 31-35 CDR2: residues 50-56 CDR2: residues 51-68 CDR3: residues95-102 CDR3: residues 99-111 Chothia CDR definition Light chain CDR1:residues 26-32 CDR1: residues 24-34 CDR2: residues 50-52 CDR2: residues50-56 CDR3: residues 91-96 CDR3: residues 89-97 Heavy chain CDR1:residues 26-32 CDR1: residues 31-35 CDR2: residues 52-56 CDR2: residues51-68 CDR3: residues 96-101 CDR3: residues 99-111

BT062 is an immunoconjugate comprising the CD138 targeting chimericantibody nBT062 that is attached via a linker, here SPDB, to thecytostatic maytansinoid derivative DM4. A chemical representation ofBT062 is provided in FIGS. 1 and 2. Immunoconjugates comprising nBT062and a maytansinoid effector molecule are often characterized in terms oftheir linker and maytansinoid effector, e.g., nBT062-SMCC-DM1, is animmunoconjugate comprising nBT062, SMCC (a “noncleavable” linkercontaining a thioester bond) and DM1 as an effector. More generically,an immunoconjugate containing nBT062 and an effector molecule may alsobe described as nBT062-linker-effector or just as nBT062-effector(nBT062N, wherein N is any effector described herein.

Reference is made herein to a unhindered counterpart (UI: unhinderedimmunoconjugate) of an immunoconjugate comprising an engineeredtargeting antibody against CD138 attached to an effector molecule via acleavable linker (CL) and is described herein as UICL, which iscontrasted to an immunoconjugate in which said effector molecule issterically hindered, and contains a cleavable linker (HICL -hinderedimmunoconjugate, cleavable linker). The UICL is an immunoconjugateequivalent to the HICL comprising an engineered targeting antibody inwhich the effector molecule is, however, not sterically hindered.Examples of a pair of HICL/UICL are BT062 and nBT062-SPP-DM1. Anunhindered counterpart of such an immunoconjugate comprising anon-cleavable linker (UINCL) refers to the equivalent immunoconjugatecomprising an engineered targeting antibody in which the effectormolecule is not sterically hindered and comprises a noncleavable linker.For BT062, nBT062-SMCC-DM1 would constitute an example of such anunhindered counterpart comprising an non-cleavable linker (UNICL).

A growth of a tumor inhibiting activity (=tumor growth inhibitingactivity) of an immunoconjugate is a relative measure. It describes thetumor growth inhibiting activity of a conjugate relative to the activityof the highest performing immunoconjugate whose activity is set as 100%.For example if the activity of the highest performing immunoconjugate,say, BT062, which causes a tumor growth delay (TGD) of 32 days, is setas 100%, the activity of, e.g., nBT062-DM1, which displays a tumorgrowth delay (TGD) of 18 days is calculated as follows:

Tumor Growth Inhibiting Activity=100×(TGD _(nBT062-DM1) /TGD _(BT062))

more generically:

Tumor Growth Inhibiting Activity=100×(TGD _(Sample) /TGD _(Reference)).

Table 3 provides suitable examples from the results depicted in FIG.11B:

TABLE 3 Tumor growth delay (TGD) and % Activity of nBT062-DMx againstMOLP-8 tumor xenografts in SCID mice based on treatment groups receivinga 450 μg/kg dose. TGD* (days) % Activity** PBS 0 0 nBT062-SMCC-DM1 18 56BT062 32 100 nBT062-SPP-DM1 13 40 *Tumor growth delay in days (TGD) asmean time in days for treatment group to reach a predetermined size (160mm³) minus the mean time for the control group to reach thispredetermined size. **Tumor Growth Inhibiting Activity = 100 ×(TGD_(Sample)/TGD_(BT062)). The activity of BT062 is defined to be 100%.

In the example provided in Table 2, BT062 provides a growth of a tumorinhibiting activity that exceeds that of its unhindered counterpart(nBT062-SPP-DM1) by 60%, and a growth of a tumor inhibiting activitythat exceeds that of its unhindered counterpart immunoconjugatecomprising a non-cleavable linker (nBT062-SMCC-DM1) by 44%.

It was previously reported that a cleavable linker in e.g.,huC242-maytansinoid immunoconjugates may provides for a so calledbystander effect. Goldmahker et al. (U.S. Patent Publication2006/0233814) also disclose that the bystander effect is particularlypronounced when the effector molecule is subject to furthermodification, in particular alkylation, upon cleavage from the targetingagent. Goldmahker et al. also showed that UICL displayed better TGD thanthe respective UINCL (see, e.g., FIG. 6 of U.S. Patent Publication2006/0233814).

However, the overall effectiveness of HICL/UICL/UINCL immunoconjugatesappear to differ from immunoconjugate to immunoconjugate and/or targetto target. For example the HICL trastuzumab-SPP-DM4 was clearlyoutperformed in its ability to reduce tumor size by the UINCLtrastuzumab-SMCC-DM1, while performance of the UICL immunoconjugatetrastuzumab-SPP-DM1 substantially resembled that of the correspondingHICL (see U.S. Patent Publication 2008/0171040 to Eberts et al.), thusrendering the results obtained a function of the immunoconjugate and thetarget.

FIG. 11A shows that the HICL outperformed the UICL and UNICL, it wasalso surprisingly found that an UICL in a high single dosage regime (250μg/kg) actually did not show any better results than the UINCL. In fact,the TGD in days that was observed in an UICL in such a regime wasactually lower than that of the UINCL. This observation became morepronounced with an increase in dosage (450 μg/kg). In sum, as shown inFIG. 11A, HICL outperformed UICL in single dose experiments to anunexpected degree. Consistent with these results, in repeat dosageexperiments, the HICL outperformed the UICL considerably, the latter ofwhich provided results which only marginally exceeded that of thecontrol. In addition, the UICL was outperformed by UINCL at higherdosages.

Adhesion of multiple myeloma cells to stromal cells, in particular bonemarrow stromal cells, has been made accountable for adhesion mediateddrug resistance (CAM-DR) that has been observed in multiple myelomapatients. In certain embodiments, the immunoconjugates of the presentinvention may alleviate CAM-DR. In particular, in certain embodiments ofthe present invention this adhesion is diminished by administering theimmunoconjugate to said multiple myeloma cells, e.g., by at least about10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%,about 80% or more. As shown in FIG. 17E, in particular UICL and HICLwere able to inhibit multiple myeloma cell adhesion to BMSCs (bonemarrow stromal cells) in samples in which just the stroma cells weretreated (as well as in samples in which the stroma cells where nottreated), while the UINCL counterpart did not have this effect. Theseresults suggest that the adhesion diminishing effect is dependent on thenature of the linker of the immunoconjugate, with cleavable linker thatallow the effector molecule to dissociate more readily, being preferred.The fact that multiple myeloma cells are at least in part prevented fromadhering to BMSCs also makes them more readily accessible to othercytotoxic agents, including those that are usually inhibited fromacting, at least with full efficiency, on the multiple myeloma cells asa result of CAM-DR. Thus, administration of the immunoconjugate ispreferably combined with a concomitant or subsequent administration ofcytotoxic agent. Time intervals between the administration of theimmunoconjugate and the cytotoxic agent may comprise 12 hours to 6 days,including 12 hours, 24 hours, 2, 3, 4 or 5 days.

The immunoconjugates disclosed herein can be administered by any route,including intravenously, parenterally, orally, intramuscularly,intrathecally or as an aerosol. The mode of delivery will depend on thedesired effect. A skilled artisan will readily know the best route ofadministration for a particular treatment in accordance with the presentinvention. The appropriate dosage will depend on the route ofadministration and the treatment indicated, and can readily bedetermined by a skilled artisan in view of current treatment protocols.

Pharmaceutical compositions containing the immunoconjugate of thepresent invention and/or any further cytotoxic agent as activeingredients can be prepared according to conventional pharmaceuticalcompounding techniques. See, for example, Remington's PharmaceuticalSciences, 17th Ed. (1985, Mack Publishing Co., Easton, Pa.). Typically,effective amounts of active ingredients will be admixed with apharmaceutically acceptable carrier. The carrier may take a wide varietyof forms depending on the form of preparation desired foradministration, for example, intravenous, oral, parenteral, intrathecal,transdermal, or by aerosol.

For oral administration, the immunoconjugate and/or cytotoxic agent canbe formulated into solid or liquid preparations such as capsules, pills,tablets, lozenges, melts, powders, suspensions or emulsions. Inpreparing the compositions in oral dosage form, any of the usualpharmaceutical media may be employed, such as, for example, water,glycols, oils, alcohols, flavoring agents, preservatives, coloringagents, suspending agents, and the like in the case of oral liquidpreparations (such as, for example, suspensions, elixirs and solutions);or carriers such as starches, sugars, diluents, granulating agents,lubricants, binders, disintegrating agents and the like in the case oforal solid preparations (such as, for example, powders, capsules andtablets). Because of their ease in administration, tablets and capsulesrepresent the most advantageous oral dosage unit form, in which casesolid pharmaceutical carriers are obviously employed. If desired,tablets may be sugar-coated or enteric-coated by standard techniques.The active agent must be stable to passage through the gastrointestinaltract. If necessary, suitable agents for stable passage can be used, andmay include phospholipids or lecithin derivatives described in theliterature, as well as liposomes, microparticles (including microspheresand macrospheres).

For parenteral administration, the immunoconjugate and/or cytotoxicagent may be dissolved in a pharmaceutical carrier and administered aseither a solution or a suspension. Illustrative of suitable carriers arewater, saline, phosphate buffer solution (PBS), dextrose solutions,fructose solutions, ethanol, or oils of animal, vegetative or syntheticorigin. The carrier may also contain other ingredients, for example,preservatives, suspending agents, solubilizing agents, buffers and thelike. When the unconjugated targeting agent and/or immunoconjugateand/or cytotoxic agent are being administered intracerebroventricularlyor intrathecally, they may also be dissolved in cerebrospinal fluid.

Dosages administered to a subject may be specified as amount, persurface area of the subject (which include humans as well as non-humananimals). The dose may be administered to such a subject in amounts,preferably, but not exclusively from about 5 mg/m² to about 300 mg/m²,including about 20 mg/m², about 50 mg/m², about 100 mg/m², about 150mg/m², about 200 mg/m² and about 250 mg/m². In certain embodiments, inwhich the immunoconjugate is administered in combination with acytotoxic agent, the dose of the immunoconjugate may be lower. Theimmunoconjugates are suitably administered at one time or over a seriesof treatments. In a multiple dose regime these amounts may beadministered once a day, once a week, once every two weeks, once everythree weeks, once every four weeks, one every five weeks or once everysix weeks. Loading doses with a single high dose or, alternatively,lower doses that are administered shortly after one another followed bydosages timed at longer intervals constitute a preferred embodiment ofthe present invention. In a preferred embodiment, the timing of thedosages are adjusted for a subject so that enough time has passed priorto a second and/or any subsequent treatment so that the previous dosehas been metabolized substantially, but the amount of immunoconjugatepresent in the subject's system still inhibits, delays and/or preventsthe growth of a tumor. An exemplary “repeated single dose” regimecomprises administering an initial dose of immunoconjugate of about 200mg/m² once every three weeks. Alternatively, a high initial dose may befollowed by a biweekly maintenance dose of about 150 μg/m². However,other dosage regimens may be useful. The progress of this therapy iseasily monitored by known techniques and assays. Dosage may varydepending on whether they are administered for preventive or therapeuticpurposes, the course of any previous therapy, the patient's clinicalhistory and response to the targeting agent/immunoconjugate, and thediscretion of the attending physician.

In one embodiment, the immunoconjugate is administered with one or moreadditional cytotoxic agents, which is efficient in treating the samedisease but which is, when administered without the immunoconjugate,often of limited effectiveness in view of CAM-DR; for example a CD138specific immunoconjugate can be administered in combination withdexamethasone. In particular, dexamethasone is administered to a patientin need thereof in effective amount two hours after the immunoconjugatehas been administered.

Thus, the immunoconjugate of the present invention may also particularlybe administered in treatment regimens with high-dose chemotherapy(preferably, melphalan, melphalan/prednisone (MP),vincristine/doxorubicin/dexamethasone (VAD), liposomaldoxorubicin/vincristine, dexamethasone (DVd), cyclophosphamide,etoposide/dexamethasone/cytarabine, cisplatin (EDAP)), stem celltransplants (e.g., autologous stem cell transplantation or allogeneicstem cell transplantation, and/or mini-allogeneic (non-myeloablative)stem cell transplantation), steroids (e.g., corticosteroids,dexamethasone, thalidomide/dexamethasone, prednisone,melphalan/prednisone), supportive therapy (e.g., bisphosphonates, growthfactors, antibiotics, intravenous immunoglobulin, low-dose radiotherapy,and/or orthopedic interventions), THALOMID (thalidomide, Celgene),VELCADE (bortezomib, Millennium), and/or REVLIMID (lenalidomide)(Chelgene Corporation) and/or other multiple myeloma treatmentsincluding radiation therapy.

If an immunoconjugate of the present invention is administered incombination with a cytotoxic agents, the above doses and regimes areoften maintained. However, if the immunoconjugate and the cytotoxicagent are co-administered, low dosages of each of these therapeuticcomponents are, in certain embodiments, preferred. In such a situation,the immunoconjugate may be administered at doses from about 5 mg/m² toabout 100 mg/m², including about 20 mg/m², about 50 mg/m², while thecytotoxic agent is administered at doses as the recommended whenadministered alone.

The experimental data obtained in the cell culture based (FIG. 7) andmouse experiments (FIGS. 8 to 11), was further confirmed withexperiments that further supported these finding.

The pathogenesis of multiple myeloma involves binding of myeloma cells,via cell-surface adhesion molecules, not only to bone marrow stromacells (BMSCs) but also extracellular matrix (ECM). This bindingtriggers, and thus can be made ultimately responsible, for multiplemyeloma cell growth, drug resistance, and migration of MM cells in thebone marrow milieu (Munshi et al. 2008). In particular, the adhesion ofmultiple myeloma cells to ECM via syndecan-1 (CD138) to type I collagen,induces the expression of matrix metalloproteinase 1, thus promotingbone resorption and tumour invasion (Hideshima et al. 2007).Interactions between multiple myeloma cells and the bone marrowmicroenvironment results in activation of a pleiotropic proliferativeand anti-apoptotic cascade.

Following the homing of multiple myeloma cells to the bone marrowstromal compartment, adhesion between multiple myeloma cells and BMSCsupregulates many cytokines like interleukin-6 (IL-6) and insulin likegrowth factor 1 (IGF-1) which have angiogenic and tumor growth promotingactivities (Hideshima et al. 2007). The signalling cascades initiated bythese cytokines eventually result in MM cell resistance to conventionaltherapeutics (Anderson et al. 2000; Hideshima et al. 2006).

The in vivo efficacy of nBT062-SPDB-DM4 and nBT062-SPP-DM1 againstCD138-positive tumor cells in the presence of human bone marrow wasanalyzed in a mouse model and the results of this analysis are shown inFIG. 12. The Figure shows that both HICL and UICL perform well in thisenvironment. The increase in the level of shulL-6R which can, in thismodel, be used as a parameter of MM cell growth, were both suppressed bythe these immunoconjugates.

In accordance with the present invention, MM is treated as follows, withthe use of BT062 as an example. This example is not intended to limitthe present invention in any manner, and a skilled artisan could readilydetermine other immunoconjugate or nBT062 based systems that are withinthe scope of the present invention and other treatment regimes whichcould be utilized for the treatment of diseases such as MM.

Due to the selective expression of CD138 on patient MM cells on via theblood stream accessible cells, the specificity of nBT062 and thestability of BT062 in the bloodstream, BT062 removes the systemictoxicity of DM4 and provides an opportunity to target the delivery ofthe DM4-effector molecule(s). The immunoconjugates of this inventionprovide a means for the effective administration of the effectormolecules to cell sites where the effector molecules can be releasedfrom the immunoconjugates.

One or more cytotoxic agents are administered in the dosages and dosageforms and according to establish treatment protocols for these cytotoxicagents to an individual that is also treated with an immunoconjugate ofthe present invention.

In particular, a patient is subjected to a treatment regime using anappropriate dosage of BT062, e.g., 100 mg/m² according to the presentinvention at certain intervals, e.g. initially daily and then weekly.Twelve hours after each weekly immunoconjugate treatment, the patient isalso treated with melphalan, e.g., by administering an oral dosageaccording to the manufacturer's instruction is administered to thepatient (e.g. a pill traded under the trademark ALKERAN).

In accordance with the present invention, in particular solid tumors mayalso be treated as follows using BT062 as an example. This example isnot intended to limit the present invention in any manner, and a skilledartisan could readily determine other immunoconjugates of the presentinvention and other treatment regimes which could be utilized for thetreatment of solid tumors. The tumor is first treated to reduce the sizeof the tumor, for example radioactively. Subsequent administration ofBT062, followed by a dose of a cytotoxic agent, e.g. in form of anALKERAN pill, provides a highly effective means for eliminating residualcancer cells. The administration of the immunoconjugate allows specifictargeting of these residual cells and release of the effector moleculesat the target site. The immunoconjugate not only overcomes or diminishesCAM-DR with respect to its own activity, but due to the fact thatinhibits multiple myeloma cell adhesion to stroma cells, theimmunoconjugate also overcomes or diminishes CAM-DR for other cytotoxicagents. The high efficacy of the immunoconjugate allows, in preferredembodiments, for a single dose regime.

The present invention is further described by reference to the followingExamples, which are offered by way of illustration and are not intendedto limit the invention in any manner. Standard techniques well known inthe art or the techniques specifically described below are utilized.

Materials and Methods

Chimeric Antibody Construction (cB-B4: nBT062)

B-B4

Murine antibody B-B4 as previously characterized (Wijdenes et al., Br JHaematol., 94 (1996), 318) was used in these experiments.

Cloning and Expression of B-B4 and cB-B4/nBT062

Standard recombinant DNA techniques were performed as described indetail in text books, for example in J. Sambrook; Molecular Cloning, ALaboratory Manual; 2nd Ed. (1989), Cold Spring Harbor Laboratory Press,USA, or as recommended by the manufacturer's instruction in the caseswhen kits were used. PCR-cloning and modification of the mouse variableregions have been conducted using standard PCR methodology. Primersindicated in the respective results section have been used.

Expression of cB-B4/nBT062

Exponentially growing COS cells, cultured in DMEM supplemented with 10%FCS, 580 μg/ml L-glutamine, 50 Units/ml penicillin and 50 μg/mlstreptomycin were harvested by trypsinization and centrifugation andwashed in PBS. Cells were resuspended in PBS to a final concentration of1×10⁷ cells/ml. 700 μl of COS cell suspension was transferred to a GenePulser cuvette and mixed with heavy and kappa light chain expressionvector DNA (10 μg each or 13 μg of Supervector). Cells wereelectroporated at 1900 V, 25 μF using a Bio-Rad Gene Pulser. Transformedcells were cultured in DMEM supplemented with 10% gamma-globulin freeFBS, 580 μg/ml L-glutamine, 50 Units/ml penicillin and 50 μg/mlstreptomycin for 72 h before antibody-containing cell culturesupernatants were harvested.

Capture ELISA to Measure Expression Levels of cB-B4/nBT062

96 well plates were coated with 100 μl aliquots of 0.4 μg/ml goatanti-human IgG antibody diluted in PBS (4° C., overnight). Plates werewashed three times with 200 μl/well washing buffer (PBS+0.1% Tween-20).Wells were blocked with 0.2% BSA, 0.02% Tween-20 in PBS, before additionof 200 μl cell culture supernatants containing the secreted antibody(incubation at 37° C. for one hour). The wells were washed six timeswith washing buffer, before detection of bound antibody with goatanti-human kappa light chain peroxidase conjugate.

Purification of cB-B4/nBT062 From Cell Culture Supernatants

The cB-B4 antibody was purified from supernatants of transformed COS 7cells using the Protein A ImmunoPure Plus kit (Pierce, Rockford, Ill.),according to the manufacturer's recommendation.

cB-B4 Binding and Competition Assay

Analysis of binding activity of B-B4 and cB-B4 to CD138 was performedusing the Diaclone (Besançon, France) sCD138 kit according to themanufacturer's recommendation, considering the changes described in theresults section.

RNA Preparation and cDNA Synthesis

Hybridoma B-B4 cells were grown and processed using the Qiagen Midi kit(Hilden, Germany) to isolate RNA following the manufacturer's protocol.About 5 μg of B-B4 RNA was subjected to reverse transcription to produceB-B4 cDNA using the Amersham Biosciences (Piscataway, N.J.) 1st strandsynthesis kit following the manufacturer's protocol.

Cloning of B-B4 Immunoglobulin cDNA

Immunoglobulin heavy chain (IgH) cDNA was amplified by PCR using the IgHprimer MHV7 (5′-ATGGGCATCAAGATGGAGTCACAGACCCAGG-3′) [SEQ ID NO:3] andthe IgG1 constant region primer MHCG1 (5′-CAGTGGATAGACAGATGGGGG-3′) [SEQID NO:4]. Similarly, immunoglobulin light chain (IgL) was amplifiedusing the three different IgK primers MKV2(5′-ATGGAGACAGACACACTCCTGCTATGGGTG-3′) [SEQ ID NO:5], MKV4(5′-ATGAGGGCCCCTGCTCAGTTTTTTGGCTTCTTG-3′) [SEQ ID NO:6] and MKV9(5′-ATGGTATCCACACCTCAGTTCCTTG-3′) [SEQ ID NO:7], each in combinationwith primer MKC (5′-ACTGGATGGTGGGAAGATGG-3′) [SEQ ID NO:8]. Allamplification products were directly ligated with the pCR2.1-TOPO vectorusing the TOPO-TA cloning kit (Invitrogen, Carlsbad, Calif.) accordingto the manufacturer's instruction.

E. coli TOP10 bacteria (Invitrogen) transformed with the ligated pCR2.1vector constructs were selected on LB-ampicillin-Xgal agar plates. Smallscale cultures were inoculated with single white colonies, grownovernight and plasmids were isolated using the QIAprep Spin Miniprep kitaccording to the manufacturer's instruction.

cDNA Sequence Determination

Plasmids were sequenced using the BigDye Termination v3.0 CycleSequencing Ready Reaction Kit (ABI, Foster City, Calif.). Each selectedplasmid was sequenced in both directions using the 1210 and 1233 primerscycled on a GeneAmp9600 PCR machine. The electrophoretic sequenceanalysis was done on an ABI capillary sequencer.

The complete cycle of RT-PCR, cloning and DNA sequence analysis wasrepeated to obtain three completely independent sets of sequenceinformation for each immunoglobulin chain.

B-B4 VK DNA Sequence

1st strand synthesis was performed in three independent reactions. ThePCR products generated by using primers MKC and MKV2 (sequences givenabove) were ligated into pCR2.1-TOPO vectors according to themanufacturer's instruction. Clones from each independent set of RT-PCRreactions were sequenced in both directions. MKV2-primed productsequence was highly similar to sterile kappa transcripts originatingfrom the myeloma fusion partner such as MOPC-21, SP2 and Ag8 (Carroll etal., Mol Immunol., 25 (1988), 991; Cabilly et al., Gene, 40 (1985); 157)and was therefore disregarded.

The PCR products using MKC with MKV4 and MKV9 primers were similar toeach other and differed only at the wobble positions within the leadersequence primer.

B-B4 VH DNA Sequence

1st strand synthesis was performed in three independent reactions andPCR products were cloned and sequenced from each 1st strand product.Five clones were sequenced from each 1st strand.

Construction of Chimeric cB-B4 Expression Vectors

The construction of the chimeric expression vectors entails adding asuitable leader sequence to VH and VK, preceded by a BamHI restrictionsite and a Kozak sequence. The Kozak consensus sequence is crucial forthe efficient translation of a variable region sequence. It defines thecorrect AUG codon from which a ribosome can commence translation, andthe single most critical base is the adenine (or less preferably, aguanine) at position -3, upstream of the AUG start. The leader sequenceis selected as the most similar sequence in the Kabat database (Kabat etal., NIH National Technical Information Service, 1991). These additionsare encoded within the forward (For) primers (both having the sequence5′-AGAGAAGCTTGCCGCCACCATGATT-GCCTCTGCTCAGTTCCTTGGTCTCC-3′ [SEQ ID NO:9];restriction site is underlined; Kozak sequence is in bold type).Furthermore, the construction of the chimeric expression vectors entailsintroducing a 5′ fragment of the human gamma1 constant region, up to anatural ApaI restriction site, contiguous with the 3′ end of the Jregion of B-B4 and, for the light chain, adding a splice donor site andHindIII site. The splice donor sequence is important for the correctin-frame attachment of the variable region to its appropriate constantregion, thus splicing out the V:C intron. The kappa intron+CK areencoded in the expression construct downstream of the B-B4 VK sequence.Similarly, the gamma-4 CH is encoded in the expression constructdownstream of the B-B4 VH sequence.

The B-B4 VH and VK genes were first carefully analyzed to identify anyunwanted splice donor sites, splice acceptor sites, Kozak sequences andfor the presence of any extra sub-cloning restriction sites which wouldlater interfere with the subcloning and/or expression of functionalwhole antibody. An unwanted HindIII site was found in the VK sequencewhich necessarily was removed by site-directed mutagenesis via PCRwithout changing the amino acid sequence. For this reactions,oligonucleotide primers BT03 (5′-CAACAGTATAGTAAGCTCCCTCGGACGTTCGGTGG-3′)[SEQ ID NO:10] and BT04 (5′-CCACCGAACGTCCGAGGGAGCTTACTATACTGTTG-3′) [SEQID NO:11] were used and mutagenesis was performed according to theStratagene (La Jolla, Calif.) Quickchange Mutagenesis Kit protocol.

Kappa Chain Chimerization Primers

The non-ambiguous B-B4 VK leader sequence, independent of the PCR primersequence, was aligned with murine leader sequences in the Kabatdatabase. The nearest match for the B-B4 VH leader was VK-10 ARS-A (Sanzet al., PNAS, 84 (1987), 1085). This leader sequence is predicted to becut correctly by the SignalP algorithm (Nielsen et al., Protein Eng, 10(1997); 1). Primers CBB4Kfor (see above) and g2258(5′-CGCGGGATCCACTCACGTTTGATTTCCAGCTTGGTGCCTCC-3′ [SEQ ID NO:12];Restriction site is underlined) were designed to generate a PCR productcontaining this complete leader, the B-B4 VK region, and HindIII andBamHI terminal restriction sites, for cloning into the pKN100 expressionvector. The forward primer, CBB4K introduces a HindIII restriction site,a Kozak translation initiation site and the VK-10 ARS-A leader sequence.The reverse primer g2258 introduces a splice donor site and a BamHIrestriction site. The resulting fragment was cloned into theHindIII/BamHI restriction sites of pKN100.

Heavy Chain Chimerization Primers

The non-ambiguous B-B4 VH leader sequence, independent of the PCR primersequence, was aligned with murine leader sequences in the Kabatdatabase. The nearest match for the B-B4 VK leader was VH17-1A (Sun etal., PNAS, 84 (1987), 214). This leader sequence is predicted to be cutcorrectly by the SignalP algorithm. Primers cBB4Hfor (see above) andg22949 (5′-CGATGGGCCCTTGGTGGAGGCTGAGGA-GACGGTGACTGAGGTTCC-3′ [SEQ IDNO:13]; Restriction site is underlined) were designed to generate a PCRproduct containing VH17-1A leader, the B-B4 VH region, and terminalHindIII and ApaI restriction sites, for cloning into the pG4D200expression vector. The forward primer cBBHFor introduces a HindIIIrestriction site, a Kozak translation initiation site and the VH17-1Aleader sequence. The reverse primer g22949 introduces the 5′ end of thegamma4 C region and a natural ApaI restriction site. The resultingfragment was cloned into the HindIII/ApaI restriction sites of pG4D200,resulting in vector pG4D200cBB4.

Production of cBB4 Antibody

One vial of COS 7 cells was thawed and grown in DMEM supplemented with10% Fetal clone I serum with antibiotics. One week later, cells (0.7 mlat 10⁷ cells/ml) were electroporated with pG4D200cBB4 plus pKN100cBB4(10 μg DNA each) or no DNA. The cells were plated in 8 ml growth mediumfor 4 days. Electroporation was repeated seven times.

Detection of Chimeric Antibody

A sandwich ELISA was used to measure antibody concentrations in COS 7supernatants. Transiently transformed COS 7 cells secreted about 6956ng/ml antibody (data not shown).

Binding Activity of cB-B4

To assay the binding activity of cB-B4 in COS 7 culture supernatants,the Diaclone sCD138 kit has been used, a solid phase sandwich ELISA. Amonoclonal antibody specific for sCD138 has been coated onto the wellsof the microtiter strips provided. During the first incubation, sCD138and biotinylated B-B4 (bio-B-B4) antibody are simultaneously incubatedtogether with a dilution series of unlabeled test antibody (B-B4 orcB-B4).

The concentrations of bio-B-B4 in this assay have been reduced in orderto obtain competition with low concentrations of unlabeled antibody(concentration of cB-B4 in COS 7 cell culture supernatants wereotherwise too low to obtain sufficient competition). Results from thisassay reveal that both antibodies have the same specificity for CD138(data not shown).

Purification of cB-B4

Chimeric B-B4 was purified from COS 7 cell supernatants using theProtein A ImmunoPure Plus kit (Pierce), according to the manufacturer'srecommendation (data not shown).

K_(D)-Determination: Comparison nBT062/BB4

Purification of Soluble CD138

Soluble CD138 antigen from U-266 cell culture supernatant was purifiedby FPLC using a 1 mL “HiTrap NHS-activated HP” column coupled with B-B4.Cell culture supernatant was loaded in PBS-Buffer pH 7.4 onto the columnand later on CD138 antigen was eluted with 50 mM tri-ethylamine pH 11 in2 mL fractions. Eluted CD138 was immediately neutralised with 375 μL 1 MTris-HCl, pH 3 to prevent structural and/or functional damages.

Biotinylation of CD138

Sulfo-NHS-LC (Pierce) was used to label CD138. NHS-activated biotinsreact efficiently with primary amino groups like lysine residues in pH7-9 buffers to form stable amide bonds.For biotinylation of CD138, 50 μl of CD138 were desalted using proteindesalting spin columns (Pierce). The biotinylation reagent (EZ-LinkSulfo NHS-LC-Biotin, Pierce) was dissolved in ice-cooled deionised H₂Oto a final concentration of 0.5 mg/mL. Biotinylation reagent and capturereagent solution were mixed having a 12 times molar excess ofbiotinylation reagent compared to capture reagent (50 pmol CD138 to 600pmol biotinylation reagent) and incubated 1 h at room temperature whileshaking the vial gently. The unbound biotinylation reagent was removedusing protein desalting columns.Immobilization of bCD138The sensorchip (SENSOR CHIP SA, BIACORE AB) used in the BIACORE assay isdesigned to bind biotinylated molecules for interaction analysis inBIACORE systems. The surface consists of a carboxymethylated dextranmatrix pre-immobilized with streptavidin and ready for high-affinitycapture of biotinylated ligands. Immobilization of bCD138 was performedon SENSOR CHIP SA using a flow rate of 10 μL/min by manual injection.The chip surface was conditioned with three consecutive 1-minuteinjections of 1 M NaCl in 50 mM NaOH. Then biotinylated CD138 wasinjected for 1 minute.

K_(D)-Determination of Different Antibodies Using BIACORE

The software of BIACORE C uses pre-defined masks, so called “Wizards”for different experiments where only certain settings can be changed. Asthe BIACORE C was originally developed to measure concentrations, thereis no wizard designed to carry out affinity measurements. However, withthe adequate settings, the wizard for “non-specific binding” could beused to measure affinity rate constants and was therefore used forK_(D)-determination. With this wizard, two flow cells were measured andthe dissociation phase was set to 90 s by performing the “Regeneration1” with BIACORE running buffer. “Regeneration 2” which is equivalent tothe real regeneration was performed with 10 mM Glycine-HCl pH 2.5. Afterthis step, the ligand CD138 was in its binding competent state again.During the whole procedure HBS-EP was used as running and dilutionbuffer. To determine binding of the different antibodies (˜150 kDa) toCD138, association and dissociation was analysed at differentconcentrations (100, 50, 25, 12.5, 6.25 and 3.13 nM). The dissociationequilibrium constants were determined by calculating the rate constantska and kd. Afterwards, the K_(D)-values of the analytes were calculatedby the quotient of kd and ka with the BIAevaluation software. Theresults are shown in Table 4.

TABLE 4 Comparative analysis of K_(D) values of nBT062 and B-B4.Standard deviations are given for mean K_(D) values. Affinity AntibodyK_(D) (nM) mean K_(D) (nM) nBT062 1.4 1.4 +/− 0.06 1.4 1.5 B-B4 1.7 1.6+/− 0.06 1.7 1.6 nBT062-SPDB-DM4 1.9 1.9 +/− 0.00 1.9 1.9 B-B4-SPP-DM12.6 2.6 +/− 0.06 2.7 2.6

Discussion

Mean K_(D) values for each antibody were calculated from threeindependent experiments. The results show that in all measurementsnBT062 exhibits slightly decreased K_(D) values compared to B-B4 (meanK_(D) values were 1.4 and 1.6 nM, respectively).

Preparation of Immunoconjugates

nBT062-DM1 and huC242-DM1

The thiol-containing maytansinoid DM1 was synthesized from the microbialfermentation product ansamitocin P-3, as previously described by Chari(Chari et al., Cancer Res. 1 (1992), 127). Preparation of humanized C242(huC242) (Roguska et al., PNAS, 91 (1994), 969) has been previouslydescribed. Antibody-drug conjugates were prepared as previouslydescribed (Liu et al., PNAS, 93 (1996), 8618). An average of 3.5 DM1molecules was linked per antibody molecule.

nBT062-DM4

BT062 is an antibody-drug conjugate composed of the cytotoxicmaytansinoid drug, DM4, linked via disulfide bonds through a linker tothe nBT062 chimerized monoclonal antibody. Maytansinoids areanti-mitotics that inhibit tubulin polymerization and microtubuleassembly (Remillard et al., Science 189 (1977), 1002). Chemical andschematic representations of BT062 (nBT062-DM4) are shown in FIGS. 1 and2.

Synthesis of DM4

DM4 is prepared from the well known derivative maytansinol (Kupchan etal., J. Med. Chem., 21 (1978), 31). Maytansinol is prepared by reductivecleavage of the ester moiety of the microbial fermentation product,ansamitocin P3, with lithium trimethoxyaluminum hydride (see FIG. 3).

DM4 is synthesized by acylation of maytansinol withN-methyl-N-(4-methydithiopentanoyl)-L-alanine (DM4 side chain) in thepresence of dicyclohexylcarbodiimide (DCC) and zinc chloride to give thedisulfide-containing maytansinoid DM4-SMe. The DM4-SMe is reduced withdithiothreitol (DTT) to give the desired thiol-containing maytansinoidDM4 (see FIG. 4 for the DM4 process flow diagram).

Immunoconjugate BT062

The procedure for the preparation of nBT062-DM4 is outlined in FIG. 5.The nBT062 antibody is modified with N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB linker) to introduce dithiopyridyl groups. DM4 is mixedwith the modified antibody at a concentration in excess of theequivalents of dithiopyridyl groups. The BT062 conjugate forms by adisulfide exchange reaction between the thiol group of DM4 and thedithiopyridyl groups introduced into the antibody via the linker.Purification by chromatography and diafiltration removes the lowmolecular weight reactants (DM4) and reaction products (thiopyridine),as well as aggregates of conjugated antibody, to produce the bulk drugsubstance.

FACS Analysis and WST Cytotoxicity Assays FACS Analysis

OPM-2 cells are plasma cell leukemia cell lines showing highlyexpressing CD138. OPM-2 cells were incubated with nBT062,nBT062-SPDB-DM4, nBT062-SPP-DM1 or nBT062-SMCC-DM1 at differentconcentrations (indicated in FIG. 6). The cells were washed andCD138-bound antibody or conjugates were detected using afluorescence-labeled secondary antibody in FACS analysis. The meanfluorescence measured in these experiments was plotted against theantibody concentration.

Cell Viability Assay

CD138⁺ MOLP-8 cells were seeded in flat bottom plates at 3000cells/well. CD138⁻ BJAB control cells were seeded at 1000 cells/well.The cells were treated with nBT062-SPDB-DM4, nBT062-SPP-DM1 ornBT062-SMCC-DM1 at different concentrations (indicated in FIG. 7) forfive days. WST reagent (water-soluble tetrazolium salt, ROCHE) was addedin order to measure cell viability according to the manufacturer'sinstruction (ROCHE). The reagent was incubated for 7.5 h on MOLP-8 cellsand for 2 h on BJAB cells. The fraction of surviving cells wascalculated based on the optical densities measured in a microplatereader using standard procedures.

Discussion

Binding of nBT062-SPDB-DM4, nBT062-SPP-DM1, nBT062-SMCC-DM1 or nBT062was analyzed by FACS. CD138⁺ OPM-2 as target cells were incubated withnBT062 or immunoconjugates and cell-bound molecules were detected usinga fluorescence-labeled secondary antibody. In FIG. 6, the meanfluorescences as measure for the amount of cell bound antibody isplotted against different antibody or conjugate concentrations. Theresults show, that nBT062-SPDB-DM4, nBT062-SPP-DM1 and nBT062-SMCC-DM1show very similar binding characteristics. In addition, the resultsstrongly suggest that the binding characteristics of the unconjugatedantibody is not affected by the conjugated toxins.

In cell viability assays, the cytotoxic activity of the antibody againstCD138⁺ MOLP-8 target cells and against CD138⁻ BJAB B-lymphoblastomacontrol cells were analyzed. Both cell lines were seeded in flat-bottomplates and incubated with increasing concentrations of theimmunoconjugates. Unconjugated antibody was used as a control. Thecytotoxic activity was analyzed five days after addition of theimmunoconjugates by using WST reagent in order to measure cellviability. In FIG. 7 (A)-(C), the fraction of surviving cells relativeto control cells treated with vehicle control is plotted againstincreasing immunoconjugate concentrations. The results show thatcytotoxic activity of nBT062-SPDB-DM4, nBT062-SPP-DM1 andnBT062-SMCC-DM1 against MOLP-8 cells is very similar. As expected,CD138⁻ BJAB control cells were not killed by the immunoconjugates,indicating that all immunoconjugates act via cell specific binding toCD138. In competition experiments, in which MOLP-8 cells werepreincubated with a molar excess of unconjugated nBT062. Preincubationsubstantially blocked the cytotoxicity of nBT062-SPDB-DM4, providingfurther evidence that the immunoconjugates kill the cells via specificbinding to CD138 onto the cell surface (FIG. 7 (D)).

Xenograft Mouse Experiments

To evaluate the importance of CD138 targeting on the anti-tumor activityof antibody-maytansinoid conjugates of a human chimeric version of theB-B4 antibody, nBT062, xenograft mouse experiments were performed. Twoversions of nBT062-maytansinoid conjugates were prepared that may differin the chemical stability of their disulfide linkages (nBT062-SPP-DM1and nBT062-SPDB-DM4). The anti-tumor activity of these antibody-drugconjugates was compared to the activity of the B-B4-SPP-DM1 conjugate(comprising the murine parental antibody), as well as unconjugated freemaytansinoid (DM4), native unmodified nBT062 antibody, and anon-targeting (irrelevant) IgG1-maytansinoid conjugate. The conjugateswere evaluated in a CD138-positive xenograft model (MOLP-8) of humanmultiple myeloma in severe combined immunodeficient (SCID) mice.

In these mice, subcutaneous tumors were established (female CB.17 SCIDmice) by inoculation with MOLP-8 cell suspensions. Treatment with asingle bolus intravenous injection was conducted when tumor volumesreached an average 113 mm³. Changes in tumor volume and body weight weremonitored twice per week. Experiments were carried out over 68 daysafter tumor cell inoculation.

Xenograft Mouse Experiments A Mice

Female CB.17 SCID mice, five weeks old, were obtained from Charles RiverLaboratories.

Human Tumor Cell Lines

MOLP-8, a human multiple myeloma cell line, was supplied from ATCC.MOLP-8 cells, which express the CD138 antigen on their cell surface anddevelop xenograft tumors in SCID mice, were maintained in RPMI-1640medium supplemented with 4 mM L-glutamine (Biowhittaker, Walkersville,Md.), 10% fetal bovine serum (Hyclone, Logan, Utah) and 1%streptomycin/penicillin, at 37° C. in a humidified atmosphere thatcontained 5% CO₂.

Part I Tumor Growth in Mice

Each mouse was inoculated with 1×10⁷ MOLP-8 cells subcutaneously intothe area under the right shoulder. The total volume was 0.2 ml permouse, in which the ratio of serum-free medium to matrigel (BDBioscience, Bedford, Mass.) was 1/1 (v/v). Prior to treatment, thexenograft tumors were monitored daily and were allowed to becomeestablished. The tumor volume reached approximately 113 mm³ about 11days after tumor cell inoculation. Tumor take rate of CB.17 SCID micewas 100%.

Eleven days after tumor cell inoculation, 42 mice were selected based ontumor volumes and body weights. The tumor volume was in a range of 68.2to 135.9 mm³. The forty-two mice were randomly divided into seven groups(A-G) of six animals each based on tumor volume.

Each of six mice in Group A received 200 μl of PBS as vehicle control.Each mouse in group B received 13.8 mg/kg of nBT062 naked antibody. Thisdose is equivalent to the amount of nBT062 antibody component in 250μg/kg of linked maytansinoid. The ratio of molecular weights ofmaytansinoids to nBT062 antibody in a conjugate molecule is approximate1/55. Each mouse in Group C received 250 μg/kg of DM4. Each mouse inGroup D received 250 μg/kg of huC242-DM4. Mice in groups E, F and Greceived 250 μg/kg of nBT062-SPDB-DM4, B-B4-SPP-DM1 and nBT062-SPP-DM1each, respectively.

All agents were intravenously administered as a single bolus injectionthrough a lateral tail vein with a 1 ml syringe fitted with a 27 gauge,½ inch needle. Prior to administration, the stock solutions of nBT062antibody, nBT062-SPDB-DM4 and nBT062-SPP-DM1 were diluted with sterilePBS to concentrations of 2 mg/ml, 28.1 μg/ml and 28.1 μg/ml,respectively, so that the injected volume for each mouse was between120-220 μl.

Part II

In a second set of experiments, MOLP-8 cells (1.5×10⁷ cells per mouse),suspended in a 50:50 mixture of serum free media and matrigel wereinjected subcutaneously in the area under the right shoulder in 100 μl.Tumor volumes reached about 80 mm³ at day 11 and the mean of thecontrols was about 750 mm³ at day 25, post cell inoculation. The tumordoubling time was estimated to be 4.58 days. Each mouse in the controlgroup (n=6) received 0.2 ml of sterile PBS administered into the lateraltail vein (i.v.) in a bolus injection. All treatment doses were based onconjugated maytansinoid. Nine groups (n=6) were treated with a singleintravenous injection of nBT062-SMCC-DM1, nBT062-SPDB-DM4, ornBT062-SPP-DM1, each at doses of 450, 250 and 100 μg/kg. An additionalgroup (n=6) received 250 μg/kg nBT062-SMCC-DM1 in a repeated dosing(weekly for five weeks). Mice were randomized into eleven groups (n=6)by tumor volume using the LabCat Program. The tumor volumes ranged from40.0 to 152.5 mm³. The mice were dosed based on the individual bodyweight.

Tumor size was measured twice per week in three dimensions using theLabCat System (Tumor Measurement and Tracking, Innovative ProgrammingAssociated, Inc., Princeton, N.J.). The tumor volume in mm³ wascalculated using the methodology described in Tomayko et al., CancerChemother. Pharmacol, 24 (1989), 148:

Volume=Length×Width×Height×½

Log₁₀ cell kill was calculated with the formula described in Bissery etal., Cancer Res., 51 (1991), 4845:

Log₁₀ cell kill=(T−C)/T _(d)×3.32

where (T−C) or tumor growth delay, is the median time in days requiredfor the treatment group (T) and the control group (C) tumors, to reach apredetermined size (600 mm³). T_(d) is the tumor doubling time, based onthe median tumor volume in the control mice, and 3.32 is the number ofcell doublings per log of cell growth.

Results

The tumor growth in individual mice is shown in FIGS. 8 and 9. The mean(+/−SD) tumor growth for each group is shown in FIG. 10.

As compared with tumor growth in the PBS-treated animals, treatment withnBT062 antibody, unconjugated free DM4 or the irrelevant non-targetingconjugate huC242-DM4 did not cause any significant inhibition of tumorgrowth.

All three CD138-targeting conjugates, nBT062-SPDB-DM4, B-B4-SPP-DM1 andnBT062-SPP-DM1, at a dose of 250 μg/kg caused marked delay in tumorgrowth. Based on the mean tumor volumes measured in the treatmentgroups, the DM4 conjugate nBT062-SPDB-DM4 was the most active one, whilethe nBT062-SPP-DM1 conjugate showed slightly increased activity ascompared to its murine counterpart B-B4-SPP-DM1 (FIG. 10). The resultsobtained in individual mice show in addition that the anti-tumoractivity obtained with B-B4-SPP-DM1 is more heterogeneously andtherefore less predicable than that measure in mice treated withnBT062-SPP-DM1. In terms of homogeneity of anti tumor activity, theother conjugate that uses nBT062 as targeting antibody nBT062-SPDB-DM4behaved similar to nBT062-SPP-DM1.

No body weight reduction was observed in any treatment group suggestingthat the treatments were well tolerated.

Discussion

The results of the analysis of three CD138-targeting conjugates inexperimental animals demonstrate the importance of targeted delivery forthe anti-tumor activity. While the maytansinoid conjugates of the humanchimeric nBT062 and the murine B-B4 antibodies show significant activityas measured by log cell kill, there was no significant impact on tumorgrowth from treatment with unconjugated DM4, unmodified native huBT062antibody, or a non-targeting control conjugate (huC242-DM4).

The immunoconjugate prepared from the human chimeric antibody,nBT062-SPP-DM1, gave slightly higher anti-tumor activity then theconjugate prepared from its murine counterpart, B-B4-SPP-DM1. Inaddition, treatment with nBT062-SPP-DM1 and nBT062-SPDB-DM4 resulted inmore homogenous responses in individual mice as compared to treatmentwith B-B4-SPP-DM1. The high binding variation of B-B4-SPP-DM1 explainedthat the measurement of the median tumor volume (+/−SD) of MOLP-8 humanmultiple myeloma xenografts in CB.17 SCID mice over time (days)post-inoculation actually provided for relatively better results forB-B4-SPP-DM1 than for nBT062-SPP-DM1 (data not shown). This feature ofimmunoconjugates using nBT062 as a targeting antibody seems to bebeneficial especially for therapeutic use of the conjugates.

Lastly, the most potent of the maytansinoid conjugates, following singleiv administration in the MOLP-8 xenograft models in SCID mice, wasnBT062-SPDB-DM4.

Bystander Killing (Cell Viability Assay)

CD138⁺ OPM2 cells and CD138⁻ Namalwa cells were seeded in round bottomplates either in separate wells or in coculture. The cells were treatedwith nBT062-SPDB-DM4 at concentrations ranging from 1×10⁻⁸ to 1×10⁻⁹ M.The fraction of viable cells was detected using WST reagent(water-soluble tetrazolium salt, ROCHE) according to the manufacturer'sinstruction (ROCHE). The fraction of surviving cells was calculatedbased on the optical densities measured in a microplate reader usingstandard procedures.

Discussion

Bystander killing of non-target cells in close proximity (as present inround bottom wells) to multiple myeloma cells upon nBT062-SPDB-DM4treatment was analysed in an in vitro study in which CD138-positive OPM2cells were cultured in coculture with CD138-negative Namawla cells (FIG.13). Generally, while CD138-positive cells are efficiently killed bynBT062-SPDB-DM4, CD138-negative cells were not affected by theconjugate. In the coculture in round bottom wells, however,nBT062-SPDB-DM4 also killed the antigen-negative cells in closeproximity to the antigen-positive cells (an effect that is oftenreferred to as bystander killing). Kovtun et al. (2006) discussed thatbystander killing mediated by maytansinoid conjugates occurs only inclose proximity to antigen-positive cells. Kovtun et al. (2006), whichis incorporated herein by reference in its entirety, also discusses theimportance of the linker of the immunoconjugate. In vivo, bystanderkilling may contribute to 1) the eradication of tumour cells thatheterogeneously express CD138, 2) the destruction of the tumourmicroenvironment by the killing of tumour stroma cells, and 3) theprevention of the selection of CD138-negative nBT062-SPDB-DM4-resistantcells.The bystander effect is of particular importance if the activity of animmunoconjugate is impaired by a target antigen that is expressed intumors in a heterogeneous fashion. If this is the case, a particularcell of a tumor expresses, if at all, the antigen not in amount thatwould allow effective direct targeting and killing of said cell by therespective immunoconjugate. The anti-tumor efficacy of nBT062-SPDB-DM4on CD138-negative cells in coculture with CD138-positive cells clarifiedthat the presence of target cells influences, under the appropriatecircumstances, the cytotoxic activity of nBT062-SPDB-DM4 towardsnon-target cells.

Xenograft Mouse Experiments B

In this set of experiments, eighty-five mice were inoculated with MOLP-8cells (1.5×10⁷ cells/mouse) subcutaneously in the right shoulder. Tumortake rate was 100%. Sixty-six SCID mice bearing bulky MOLP-8 tumors witha mean tumor volume of about 80 mm³ were randomized into eleventreatment groups (n=6). Mice were treated with a single dose of one ofthree conjugates (nBT062-SMCC-DM1, nBT062-SPDB-DM4 or nBT062-SPP-DM1).An additional group received five weekly doses of nBT062-SMCC-DM1 and acontrol group received a single dose of PBS. Mean tumor volumes areshown in FIG. 11A. A dose response was established for each conjugate. Amedian tumor volume of 750 mm³ in the PBS-treated animals was reached onday 25. Tumor doubling time determined by the best-fit linear regressioncurve fit on a log-linear plot of control tumor growth was 4.58 days.Animals treated with nBT062-SPDB-DM4 at 450 μg/kg had the highest logcell kill (LCK=2.89), followed by animals treated with nBT062-SMCC-DM1at 250 μg/kg weekly dosing (LCK=2.1; see Table 5). Comparison of themean tumor growth curves for the treatment groups by repeated measuresANOVA performing Dunnett's Multiple Comparison Test showed a significantdifference between the PBS control group and 450 μg/kg nBT062-SPDB-DM4(p<0.01), 250 μg/kg nBT062-SPDB-DM4 (p<0.05) and five weekly doses of250 μg/kg nBT062-SMCC-DM1 (p<0.05). No partial or complete tumorregression in any of the treatment groups occurred with the exception ofone animal receiving 450 μg/kg nBT062-SPDB-DM4, which had partialregression of the tumor until day 85 post-inoculation.

TABLE 5 Log cell kill (LCK) values as measure for anti-tumor activity ofdifferent nBT062-DMx conjugates in different dosing schemes. Refer tothe Materials and methods section for information on calculation of LCKvalues. Test Material Dose (μg/kg) LCK Dosing PBS single dosenBT062-SMCC-DM1 450 0.85 single dose nBT062-SMCC-DM1 250 0.53 singledose nBT062-SMCC-DM1 100 0 single dose nBT062-SPDB-DM4 450 2.89 singledose nBT062-SPDB-DM4 250 1.05 single dose nBT062-SPDB-DM4 100 0.39single dose nBT062-SPP-DM1 450 0.8 single dose nBT062-SPP-DM1 250 0.39single dose nBT062-SPP-DM1 100 0.2 single dose nBT062-SMCC-DM1 250 2.1weekly for 5 weeksIn Vivo Efficacy of nBT062-SPDB-DM4 and nBT062-SPP-DM1 in the BoneMarrow Environment

Preparation of SCID Mice Having Human Fetal Bone Implants

Human fetal long bones (human fetal bone chips) were implanted into theupper body of CB17 SCID-mice (SCID-hu) as previously described (Urashimaet al., 1997) and thus provided for a model in mouse for the homing ofhuman MM cells to human BM cells.

Treatment Regime (SCID-hu/INA-6 mice)4 weeks following bone implantation, 2.5×10⁶ INA-6 cells in a finalvolume of 100 μL RPMI-1640 cell culture medium were injected directlyinto the human bone marrow cavity in the SCID-hu mice described above.An increase in the levels of soluble human IL-6 receptor (shulL-6R),which is released by INA-6 cells, was used as a parameter of MM cellgrowth and disease burden.Mice developed measurable serum shulL-6R approximately 4 weeks followingINA-6 cell injection and then received 0.176 mg conjugate or vehiclecontrol via tail vein injection weekly for 7 weeks. After eachtreatment, blood samples were collected and measured for shulL-6R levelsby an enzyme-linked immunosorbent assay (ELISA, R&D Systems,Minneapolis, Minn.). The results are depicted in FIG. 12.

Discussion

Interleukin 6 (IL-6) is a growth and survival factor for multiplemyeloma cells. INA-6 is an IL-6-dependent human myeloma cell line, whichalso requires bone marrow stromal cells (BMSC) to proliferate. INA-6cell lines produce soluble IL-6 receptor (shulL-6R). An increase in thelevels of shulL-6R can be used as a parameter of MM cell growth anddisease burden.

Thus, the sCID-hu/INA-6 mice provide a model for multiple myeloma cellsgrowing in their normal bone marrow environment. The tumor cells of thismodel, which directly interact with the human bone marrow, closelyresemble the situation in patients, in which tumor cell growth is alsopromoted by the presence of stromal cells. As INA-6 cells releasesoluble human interleukin-6 receptor (shulL-6R), serum concentrations ofthis protein can be used as a measure for tumor cell load in these mice.The in vivo potency of nBT062-SPDB-DM4 and nBT062-SPP-DM1 were tested inthis environment.

Treatment of SCIDhu/INA-6 mice with weekly i.v. administrations ofnBT062-SPDB-DM4 or nBT062-SPP-DM1 for seven weeks induced efficienttumour regression, as detected by a decrease in serum shulL-6R levelsrelative to the control, indicating good efficacy of the conjugates evenin the environment of human bone marrow, which reflect the relevantsituation in patients (FIG. 12).Analysis of the Efficacy of nBT062-SMCC-DM1, nBT062-SPDB-DM4 andnBT062-SPP-DM1 in Vitro and in Experimental Animals in Vivo.

Materials and Methods Cell Culture

Dex-sensitive (MM.1 S) and resistant (MM.1 R) human MM cell lines werekindly provided by Dr. Steven Rosen (Northwestern University, Chicago,Ill.). RPM18226 and U266 human MM cell lines were obtained from AmericanType Culture Collection (Manassas, Va.). Doxorubicin (Dox)-resistant(RPMI-Dox40) and Melphalan-resistant (LR5) cells were kindly provided byDr. William Dalton (Lee Moffitt Cancer Center, Tampa, Fla.). OPM1 andOPM2 plasma cell leukemia cells were kindly provided by Dr. EdwardThompson (University of Texas Medical Branch, Galveston).

All MM and bone marrow (BM) stroma cell lines were cultured inDulbecco's modification of Eagle's medium DMEM (Sigma) containing 10%fetal bovine serum, 2 mM L-glutamine (Life Technologies), 100 U/mLpenicillin and 100 Ag/mL streptomycin (Life Technologies). Blood samplescollected from healthy volunteers were processed by Ficoll Paquegradients to obtain peripheral blood mononuclear cells (PBMCs).Patient-derived MM and BM cells were obtained from BM samples afterinformed consent was obtained per the Declaration of Helsinki andapproval by the Institutional Review Board of the Dana-Farber CancerInstitute (Boston, Mass.). BM mononuclear cells were separated usingFicoll Paque density sedimentation, and plasma cells were purified (>95%CD138⁺) by positive selection with anti-CD138 magnetic activated cellseparation micro beads (Miltenyi Biotec Auburn, Calif.). Tumor cellswere purified from the BM of patients with MM using the RosetteSepnegative selection system (StemCell Technologies, Vancouver, BC,Canada), as described previously (Hideshima, 2001). RosetteSep antibodycocktail was given to Bone marrow samples. CD138 negative cells werecrosslinked to red blood cells (rosetted) with RosetteSep reagents, andincubated for 20 minutes at room temperature, prior to separation byFicoll density centrifugation.

Growth Inhibition and Proliferation Assay

The growth inhibitory effect of nBT062-SMCC-DM1, nBT062-SPDB-DM4,nBT062-SPP-DM1 and Dexamethasone on growth of MM cell lines, PBMCs, andBMSCs was assessed in MTT-assays by measuring3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetra-sodium bromide (MTT;Chemicon International, Temecula, Calif.) dye metabolization, asdescribed previously (Hideshima, 2003).Effect of nBT062-SMCC-DM1, nBT062-SPDB-DM4 and nBT062-SPP-DM1 on MM CellGrowth in the BMTo evaluate the growth stimulatory effect of BM cells on the sensitivityof MM cells towards immunoconjugates, MM cells (2×10⁴ cells/well) wereco-cultured for 48 h with bone marrow stromal cells (BMSCs, 1×10⁴cells/well) in 96-well plates (Costar, Cambridge, Mass.), in thepresence or absence of drug. DNA synthesis was measured by[3H]-thymidine (Perkin-Elmer, Boston, Mass.) uptake. [3H]-thymidine (0.5μCi/well) was added during the last 8 hours of the experiment. Allexperiments were performed in quadruplicates.

Cell Cycle Analysis

MM cells (1×10⁶ cells) were incubated for 48 hours in presence orabsence of immunoconjugates, washed with phosphate-buffered saline(PBS), permeabilized by a 30-minute exposure to 70% ethanol at −20° C.,incubated with propidium iodide (PI) (50 μg/mL) in 0.5 mL PBS containing20 U/mL RNAse A (Roche Diagnostics) for 30 minutes at room temperature.DNA-content was analyzed using a flow cytometer.

Immunofluorescence

Cells grown on glass cover slips were fixed in cold absolute acetone for10 min, washed in PBS and blocked for 60 min with 5% FBS in PBS. Slideswere then incubated with anti-CD138 antibody (sc12765, Santa CruzBiotechnology, Santa Cruz, Calif.) for 24 h at 4° C. Cells were againwashed with PBS and incubated with fluorescence labeled goat anti-mouseIgG for 1 h at 4° C. and analyzed using a Nikon E800 fluorescencemicroscope as described previously (Ikeda, 2005; Kiziltepe, 2007).

Auto Fluorescent Green Fluorescent Protein-Positive (GFP) Human MmXenograft Model and Human MM Xenograft Murine Model.

OPM1 cells were transfected with green fluorescent protein (OPM1^(GFP))using a lentiviral vector, as previously described (Zufferey, 1998).CB17 SCID mice (48-54 days old) were purchased from Charles RiverLaboratories (Wilmington, Mass.). All animal studies were conductedaccording to protocols approved by the Animal Ethics Committee of theDana-Farber Cancer Institute. The mice were inoculated subcutaneously inthe right flank with 5×10⁶ OPM1^(GFP) MM cells in 100 μl RPMI-1640. Whentumors were palpable, mice were assigned into the treatment groupreceiving 176 μg/mouse based on the molecular weight of the conjugate,once weekly for 4 weeks by lateral tail vein injection, and 5 mice wereassigned into the control group receiving vehicle alone. Calipermeasurements of the longest perpendicular tumor diameters were performedevery other day to estimate the tumor volume using the following formularepresenting the 3D volume of an ellipse: 4/3 ×(width/2)²×(length/2).Animals were killed when tumor reached 2 cm or if the mice appearedmoribund. Survival was evaluated from the first day of treatment untildeath. Tumor growth was evaluated using caliper measurements from thefirst day of treatment until day of killing which was day 10 for controland day 21 for the BT062-SPDB-DM4 treatment group. Mice were monitoredby whole-body fluorescence imaging using a Illumatool Bright LightSystem LT-9900 (Lightools Research, Encinitas, Calif.), following acutaneous shave of the tumor area. Images were captured with a canon IXYdigital 700 camera. Ex vivo analysis of tumor images were captured witha LEICA DM IL microscope connected to a LEICA DFC300 FX camera at 40u/0.60 (Leica, Heidelberg, Germany).

Detection of Apoptotic Cells

MM cells (1×10⁶) were washed with PBS and incubated in the presence orabsence of immunoconjugates. Apoptotic cells were stained withPE-conjugated Apo 2.7 antibody (7A6, Beckman Coulter, Inc.). The cellswere analyzed by flow-cytometry on an Epics flow cytometer (BeckmanCoulter, Inc.) using the on RXP Cytomics software.

Western Blotting

MM cells were cultured in the presence or absence of nBT062-SMCC-DM1,nBT062-SPDB-DM1 or BT062-SPP-DM1, harvested, washed and lysed usingradioimmuno precipitation assay (RIPA) buffer containing 2 mM Na₃VO₄, 5mM NaF, 1 mM phenylmethylsulfonyl fluoride, 5 mg/ml Complete proteaseinhibitor, as described previously (Yasui, 2005; Hayashi, 2002).Whole-cell lysates (20 μg per lane) were subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation,transferred to Pure Nitrocellulose membranes (Bio-Rad Laboratories,Hercules, Calif.) and immunoblotted with antibodies against poly-ADP(adenosine diphosphate)-ribose polymerase (PARP), caspase-8, caspase-3,caspase-9, AKT, and phospho(Ser 473) Akt (Cell Signaling), as well asanti-tubulin and anti-CD138 antibodies (Santa Cruz Biotechnology).

Cell Adhesion Assay

In a 96-well plate, 1×10⁴ BMSCs were pipetted into each well andincubated for 12 h in the presence or absence of immunoconjugates at 37°C. Upon that incubation, MM cells in the presence or absence of drugswere given to the BMSCs. Therefore, MM cells were washed 3 times withPBS and resuspended in serum-free RPMI medium at cell densities of 2×10⁵cells in 100 μL in the presence or absence of drugs. Each sample groupwas run in either triplicate or quadruplicate. After 2 hours incoculture at 37° C., non-adherent, weakly adhered cells and medium wereremoved by inverting the plate. Subsequently, the wells were washedthree times with PBS. The remaining adherent cells were cultured in 10%FBS in RPMI medium in the presence of [3H]-thymidine (0.5 μCi/well,Perkin Elmer, Boston, Mass., USA) for further 8 hours, to measure DNAsynthesis.

Statistical Analysis

Statistical significance of differences observed in immunoconjugate(IC)-treated versus control cultures was determined using the Dunn'smultiple comparison tests. The minimal level of significance was a Pvalue less than 0.5. For in vivo experiments, tumor volumes werecompared using 1-way analysis of Dunn's multiple comparison tests.Survival was assessed using Kaplan-Meier curves and log-rank analysis.

Results Expression of CD138 on MM Cell Lines

The expression levels of CD138 in multiple myeloma cell lines by Westernblotting using whole cell lysates of MM1S, OPM1, OPM2, RPM18226, DOX40,MM1R, LR5, and U266 cells were analyzed (FIG. 14A)As can be seen in FIG. 14A, MM1S and LR5 showed week expression of CD138and Dox40 cells are CD138 negative. The other cell lines showed highexpression levels of CD138. It was shown that CD138 is expressed in 7out of 8 (87.5%) multiple myeloma cell lines.FIG. 14B shows an analysis using immunofluorescence staining using CD138specific antibodies. Microscopic analysis of DOX40 cells (upper panel)and OPM1 cells (lower panel) was performed. CD138 expression is shown onthe left and nucleic acids on the right. FIG. 14B shows that DOX40 cellsdisplay almost no detectable expression of CD138. In contrast, OPM1cells showed high CD138 immunoreactivity.nBT062-SMCC-DM1, nBT062-SPDB-DM4 and nBT062-SPP-DM1 Show SelectiveCytotoxicity to CD138 Positive Cell LinesThe efficacy of CD138 antibody maytansinoid conjugates were also testedin cell viability assays. The immunoconjugates nBT062-SMCC-DM1,nBT062-SPDB-DM4 and nBT062-SPP-DM1 were assayed for their cytotoxicpotency against CD138-positive cells (OPM1, RPM18226), CD138 weaklyexpressing cells (MM1S) and CD138-negative cells (DOX40), using a3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT)-basedassay. FIG. 15A depicts OPM1 (▪), RPM18226 (♦), DOX40 (□) and MM1S cells(Δ) as exposed to nBT062-SMCC-DM1, nBT062-SPDB-DM4 or nBT062-SPP-DM1,respectively. Cell survival was measured in MTT assays. Cell viabilitiesafter indicated incubation times (40, 80, 120 h) are given in % ofviability of untreated controls. Treatment of the cells withnBT062-SMCC-DM1, nBT062-SPDB-DM4 and nBT062-SPP-DM1 at concentrationsranging from 3 to 354 ng/ml induced growth inhibition in CD138 positivecells. This effect occurred in a time and dose dependent manner and wasmost prominent after 120 h in the two cell lines expressing high levelsof CD138. Under the same conditions, almost no cytotoxicity could bemeasured against CD138 negative DOX40 cells (FIG. 15A). Importantly, thethree immunoconjugates were also cytotoxic against patient-derivednegative selected MM cells as analyzed in a concentration range of111-442 ng/ml, measured after an incubation time of 48 h (FIG. 15B).FIG. 15C shows PBMCs derived from 3 healthy subjects that were culturedwith nBT062-SPDB-DM4 for 72 h before cell viability was determined. Cellviability of OPM1 mM treated under the same conditions is shown as acontrol (closed squares). As can be seen from FIG. 13 15C, theimmunoconjugates did not induce cytotoxic effects in PBMCs (PeripheralBlood Mononuclear Cells) isolated from 3 healthy volunteers (FIG. 15C).These results demonstrate that nBT062-SMCC-DM1, nBT062-SPDB-DM4 andnBT062-SPP-DM1 selectively kill CD138-positive MM cells (All data shownin this figure represent averages of triplicates. Standard deviationsare indicated by error bars).nBT062-SMCC-DM1, nBT062-SPDB-DM4 and nBT062-SPP-DM1 Induce G2/M CellCycle Arrest in OPM1 CellsAs maytansinoid acts by suppressing microtubule polymerization, it haspreviously been shown that DM1 and DM4 induces G2/M cell cycle arrest intumor cells (Erickson, 2006). Thus, the cell cycle profile of OPM1 cellswere examined after treatment with nBT062-SMCC-DM1, nBT062-SPDB-DM4 andnBT062-SPP-DM1. Cells were incubated with the immunoconjugates for 0 to72 hours, labeled with PI, and analyzed by flow-cytometry. FIG. 16Ashows OPM1 cells treated with immunoconjugates for 0, 12 or 24 h and theanalysis of cell cycle profiles by PI staining. As can be seen from FIG.16A, these compounds had significant effects on the proportion of cellsin G2/M-phase as compared to untreated cells. Exposure of OPM1 cells tonBT062-SPDB-DM4 induced a quicker and stronger response compared tocells treated with the other two drugs.nBT062-SPDB-DM4 Induces Apoptosis in OPM1 CellsNext, it was determined whether nBT062-SPDB-DM4 induces apoptosis intarget cells. FIG. 16B shows OPM1 cells cultured in the presence orabsence of immunoconjugates for 24, 48 or 72 h. The percentage ofapoptotic cells was assessed by Apo 2.7 antibody staining andflow-cytometric analysis. As shown in FIG. 16B, the staining of thecells with the Apo2.7 antibody demonstrated apoptosis induction of cellstreated with the immunoconjugates.

FIG. 16C shows OPM1 cells cultured in the presence of 885 μg/mlnBT062-SPDB-DM4 for the indicated times (left panel) or with differentconcentrations of immunoconjugate (middle panel). In this Figure, FLindicates bands corresponding to full-length forms of caspases and PARP;CL indicates bands corresponding to the cleavage products. OPM1 cellswere pre-incubated with zVAD-fmk (50 μmol/L) for 60 minutes prior totreatment with nBT062-SPDB-DM4 for 24 h at indicated doses. Total celllysates were subjected to immunoblotting using caspase-3, -8, -9, PARP,and tubulin specific antibodies. As can be seen from FIG. 16C, treatmentof OPM1 cells with nBT062-SPDB-DM4 induced cleavage of caspase-8, -9,and -3 and PARP in a dose and time-dependent fashion (left and middlepanel). The pan-caspase inhibitor zVAD-fmk blocked nBT062-SPDB-DM4induced caspase-3, -8, -9 and PARP cleavage in OPM1 cells (FIG. 16C,right panel). These results indicate that nBT062-SPDB-DM4 activatescaspases and induces apoptosis in target cells.

nBT062-SMCC-DM1, nBT062-SPDB-DM4 and nBT062-SPP-DM1 Inhibit theProtective Effect Of BMSCsIn multiple myeloma patients, the bone marrow microenvironment inducesgrowth, survival, and drug resistance in MM cells via at least twodifferent mechanisms: adhesion of MM cells to fibronectin confers celladhesion-mediated drug resistance (CAM-DR); and cytokines asinterleukin-6 (IL-6) and insulin like growth factor 1 (IGF-1) in thebone marrow milieu induce important signalling cascades that finallymediate MM cell resistance to conventional therapeutics. Cytotoxicity ofthe immunoconjugates towards MM cells in co-culture with BMSCs wasanalyzed in the presence of IL-6 or IL-6 or in the presence of bonemarrow stromal cells (BMSCs).In FIGS. 17A to 17C, OPM1 cells were cultured for 48 h with controlmedia (black bars) or with increasing concentrations of nBT062-SPDB-DM4:55 ng/ml (dark gray bars; *), 111 ng/ml (gray bars; **), 221 ng/ml(light gray bars; ***), 442 ng/ml (very light gray bars; ****) 885 ng/ml(white bars; *****). Cells in FIG. 17D were treated for 72 h withcontrol media (black bars) or with increasing concentrations ofDexamethasone: 250 nM (grey bars: +), 500 nM (light grey bars; ++), 1000nM (white bars; +++). IL-6 was present in some cultures atconcentrations of 1 or 10 ng/ml (FIG. 17A). IGF-1 was used atconcentrations of 10 or 50 ng/ml (FIG. 17B). None of these cytokinesinduced resistance of the cells towards the immunoconjugates.To analyze the influence of the BM microenvironment on growth andresistance of OPM1 towards the immunoconjugates, cells were cultured inthe presence or absence of BMSCs as described above.

Coculturing of OPM1 cells in the presence or absence of BMSCs is shownin panel (C) and (D) of FIG. 17. In all experiments, DNA synthesis wasdetermined by measuring [3H]-thymidine incorporation during the last 8 hof culture. Adherence of OPM1 cells to BMSCs triggered increased[3H]-thymidine uptake. Importantly, cytotoxicity of the immunoconjugateswas not affected by the presence of BMSCs (FIG. 17C). In contrast,Dexamethasone, which was included as a control, was not able to overcomethe BMSC triggered protective effect (FIG. 17D).

nBT062-SPDB-DM4 and nBT062-SPP-DM1 Inhibit MM1s Cell Adherence to BMSCsIt was analyzed whether the immunoconjugates inhibit multiple myelomacell adhesion to BMSCs. MM1S cells, which express only moderate levelsof CD138 and showed only weakly sensitivity towards BT062 (FIG. 14A andFIG. 15A) were cultured with or without nBT062-SMCC-DM1, nBT062-SPDB-DM4or nBT062-SPP-DM1 for 2 h (885 ng/ml). After that treatment, MM1S cellswere cocultured with BMSCs for further 2 hours. In some samples, theBMSCs were also treated with the immunoconjugates for 12 h (885 ng/ml)prior to coculturing (stroma treat). After 3 washing steps with PBS,adhering cells were measured by [3H]-thymidine uptake.FIG. 17E shows BMSCs cultured 24 hours in 96 well flat bottom plates inthe presence or absence of immunoconjugates. BMSCs were washed threetimes with PBS. MM1 S cells which were incubated for 2 hours with theimmunoconjugates were given to the BMSCs. DNA synthesis was measured by[3H]-thymidine uptake.As depicted in FIG. 17E, nBT062-SPDB-DM4 and nBT062-SPP-DM1 inhibitedmultiple myeloma cell adhesion to BMSCs compared to samples in whichjust the stroma cells were treated (3.6 fold and 2.5 fold,respectively). nBT062-SMCC-DM1 showed almost no effect on cell adhesion.The result suggest that nBT062-SPDB-DM4 and nBT062-SPP-DM1 are able todisturb MM cell adhesion to BMSCs and that these immunoconjugates arealso able to overcome cell adhesion-mediated drug resistance (CAM-DR).nBT062-SPDB-DM4 Inhibits Tumor Growth in a Human Mm Xenograft Model inSCID MiceThe in vivo activities of nBT062-SPDB-DM4 and nBT062-SPP-DM1 weredetermined in a GFP-positive human MM xenograft model in SCID mice.FIG. 18A shows an analysis of GFP expression by OPM1^(GFP) cells. Asdepicted in the Figure, a highly fluorescent clone of OPM1 mM cells(OPM1^(GFP)) could be established and the anti tumor activity of theimmunoconjugates with these cells in experimental animals in vivo wereexamined. In FIG. 18B, 5 mice per group were injected with 5×10⁶OPM1^(GFP) cells. The mice were inoculated subcutaneously with 5×10⁶OPM1^(GFP) cells in 100 μl RPMI-1640 media. Treatment withnBT062-SPDB-DM4 (squares), nBT062-SPP-DM1 (triangles) or buffer controlwas begun when tumors were established. Tumor sizes were determined byserial caliper of perpendicular diameters. Error bars indicate standarddeviations. All mice developed measurable tumors 14 days after injectionof tumor cells and were then randomized to receive treatment once weeklywith nBT062-SPDB-DM4, nBT062-SPP-DM1 or control vehicle (PBS). Serialcaliper measurements of perpendicular diameters were performed everyother day to calculate tumor volume. Treatment of tumor-bearing micewith nBT062-SPDB-DM4 (176 μg/mouse based on the molecular weight of theconjugate, once weekly for 4 weeks) significantly inhibited MM tumorgrowth, as compared with control animals treated with PBS vehicle(Dunn's multiple comparison test; control vehicle vs. nBT062-SPDB-DM4:P<0.01 FIG. 16B). The nBT062-SPP-DM1 conjugate was less effective thannBT062-SPDB-DM4 (Dunn's multiple comparison test; nBT062-SPP-DM1 vs.BT062-SPDB-DM4: P<0.05 FIG. 5B). All control mice had to be killed atday 19 after begin of treatment, because of large tumor sizes. UsingKaplan-Meier curves and log-rank analysis, the mean OS was 13.6 days(95% confidence interval [CI], 10-19 days) in the control cohort versus26 days (95% CI, 23-42 days) in groups treated with nBT062-SPDB-DM4,respectively (FIG. 5C). As can be seen from FIG. 18C, nBT062-SPDB-DM4significantly increased survival (P<0.0023, dashed line, n=5) comparedwith the control group treated with vehicle only (solid line; normalsaline, n=5). Mice were killed and tumors from a nBT062-SPDB-DM4 treatedmouse and a buffer treated control mouse were excised for TUNELanalysis. Ex vivo analysis of tumors excised from OPM1^(GFP)-bearingmice showed significantly increased apoptosis in BT062-treated animalsas compared to control mice (FIG. 18D). Thus, nBT062-SPDB-DM4 inducesapoptosis in vivo. Neither of the compounds administered showed anyinfluence on body weight in this study.

Discussion

Above, a selective anti tumor activity of nBT062-SMCC-DM1,nBT062-SPDB-DM4 and nBT062-SPP-DM1 against CD138 expressing MM cells invitro and in experimental animals in vivo was demonstrated. Usingimmunoblotting and immunofluorescence analysis, we found that themajority of MM cell lines analyzed express CD138. DOX40, however, didnot express CD138 protein and MM1S and LR5 cells showed weak expressionof the protein. The remaining five showed high levels of CD138expression. The immunoconjugates nBT062-SMCC-DM1, nBT062-SPDB-DM4 andnBT062-SPP-DM1 showed significant cytotoxicity against MM cell lineswith nBT062-SPDB-DM4 being the most potent compound of these threeagents. OPM1 and RPM18226 cells which express high levels of CD138 weremore sensitive towards the immunoconjugates than CD138 low expressingMM1s cells or CD138 negative Dox40 cells. Importantly, these agents werealso cytotoxic against tumor cells isolated from patients suffering fromMM. Importantly, no cytotoxicity was observed against peripheral or bonemarrow mononuclear cells from healthy volunteers. These results suggestthat the immunoconjugates have antigen-selective activity against MMtumor cells. It could be demonstrated that nBT062-SMCC-DM1,nBT062-SPDB-DM4 and nBT062-SPP-DM1 inhibit the cell proliferation of MMcells by inducing G2/M cell cycle arrest leading to apoptotic celldeath. Cleavage of caspase-3, -8, -9 and the caspase-3 downstream targetPARP can be detected in OPM1 cells treated with the immunoconjugates. Inaddition, APO2.7 antigen -positive cells were increased aftertwenty-four hours incubation with these drugs.It was previously reported that IL-6 triggers proliferation of multiplemyeloma cells via activation of PI3-K/Akt, MEK/ERK and JAK2/STAT3signaling cascades. IGF-1 has been described to also promote multiplemyeloma cell proliferation and survival using the same pathways. IL-6protects against dexamethasone-induced apoptosis via induction ofPI3-K/Akt signaling. It was examined whether exogenous IL-6 and IGF-1inhibit immunoconjugate-induced cytotoxicity in multiple myeloma cells.Although increased proliferation was noted in cells treated with IL-6 orIGF-1, these cytokines could not inhibit nBT062-SPDB-DM4-inducedapoptosis in OPM1 cells, suggesting that these agents can overcome theprotective effects of these cytokines. Importantly, the immunoconjugatesinhibited growth and adherence of MM1S cells adhered to BMSCs, furtherconfirming that they can overcome cell adhesion-mediated drug resistance(CAM-DR). In experimental animals, the nBT062-SPDB-DM4 inducedsignificant growth delay of established MM xenografts withoutinfluencing mice body weights.All conjugates tested showed high cytotoxic activity against MM celllines and against patient-derived primary MM cells, whereas peripheralblood mononuclear cells from healthy volunteers showed no sensitivitytowards the conjugates.The CD138 specific immunoconjugates triggered G2/M cell cycle arrest andinduced apoptosis in target cells, associated with cleavage ofcaspase-8/-9/and -3 and cleavage of the caspase-3 downstream targetPARP. Importantly, interleukin-6, insulin-like growth factor-I orpresence of bone marrow stromal cells did not protect MM cells againstimmunoconjugate mediated cytotoxicity.The nBT062-SPDB-DM4 conjugate significantly inhibited MM tumor growth(p<0.01) and prolonged survival of the mice.

It will be appreciated that the methods and compositions of the instantinvention can be incorporated in the form of a variety of embodiments,only a few of which are disclosed herein. It will be apparent to theartisan that other embodiments exist and do not depart from the spiritof the invention. Thus, the described embodiments are illustrative andshould not be construed as restrictive.

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1. A method for diminishing adhesion of stroma cells to CD138 expressingtumor cells in tumor cells of a subject in need thereof comprising:administering to said tumor cells an immunoconjugate targeting saidCD138 expressing tumor cells in an amount effective to diminish adhesionof stroma cells to CD138 expressing tumor cells, and optionallyadministering to said tumor cells a further cytotoxic agent in a growthof tumor cells inhibiting, delaying and/or preventing amount.
 2. Themethod of claim 1, wherein said immunoconjugate comprises an effectormolecule and a targeting agent, wherein said effector molecule and saidtargeting agent are attached to each other via a cleavable linker. 3.The method of claim 2, wherein the cleavable linker contains a disulfidebond.
 4. The method of claim 3, wherein the linker is SPP or SPDB. 5.The method of any of claims 1 to 4, wherein the adhesion is diminishedby at least about 10%, about 20%, about 30%, about 40%, about 50%, about60%, about 70%, about 80% or more.
 6. The method of claims any of claims1 to 4, wherein said diminished adhesion results in alleviation ofadhesion mediated drug resistance.
 7. The method of claim 6, whereinadhesion mediated drug resistance against a further cytotoxic agent thatis not said immunoconjugate is diminished and wherein said furthercytotoxic agent is administered in a growth of tumor cells inhibiting,delaying and/or preventing amount.
 8. The method of claim 7, whereinsaid cytotoxic agent is mephalan, vincristine, doxorubicin,dexamethasone, cyclophosphamide, etoposide, cytarabine, cisplatin,thalidomide, prednisone, thalidomide, bortezomib, lenalidomide,sorafenib, romidepsin or combinations thereof.
 9. The method of claim 7,wherein the cytotoxic agent is antibody based.
 10. The method of claims1 or 2, wherein said effector molecule is sterically hindered.
 11. Themethod of claim 1 or 2, wherein the effector molecule is at least onemaytansinoid, taxane or a CC1065, or an analog thereof.
 12. The methodof claim 11, wherein the effector molecule is at least one maytansinoidsuch as DM1, DM3, or DM4.
 13. The method of claim 12, wherein saideffector molecule is DM4.
 14. The method of any of claims 1 to 4,wherein said targeting agent of the immunoconjugate comprises: an aminoacid sequence of an immunoglobulin heavy chain or part thereof, whereinsaid immunoglobulin heavy chain or part thereof has at least 70%, atleast 80%, at least 90%, at least 95% or least 98% sequence identitywith SEQ ID NO:1.
 15. The method of claim 7, wherein the immunoconjugateand the cytotoxic agent are administered consecutively, whereinadministration of the cytotoxic agent follows the administration of theimmunoconjugate.
 16. The method of claim 7, wherein the immunoconjugateand the cytotoxic agent are co-administered.
 17. The method of any ofclaims 1 to 4 and 7 to 9, wherein said subject is a cancer patientsuffering from one of the following: multiple myeloma, ovariancarcinoma, kidney carcinoma, gall bladder carcinoma, breast carcinoma,prostate cancer, lung cancer, colon carcinoma, Hodgkin's andnon-Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), acutelymphoblastic leukemia (ALL), acute myeloblastic leukemia (AML), a solidtissue sarcoma or a colon carcinoma.
 18. The method of claim 17, whereinsaid patient is suffering from multiple myeloma.